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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.
J Cell Sci ; 133(4)2020 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-32005696

RESUMO

USP16 (also known as UBP-M) has emerged as a histone H2AK119 deubiquitylase (DUB) implicated in the regulation of chromatin-associated processes and cell cycle progression. Despite this, available evidence suggests that this DUB is also present in the cytoplasm. How the nucleo-cytoplasmic transport of USP16, and hence its function, is regulated has remained elusive. Here, we show that USP16 is predominantly cytoplasmic in all cell cycle phases. We identified the nuclear export signal (NES) responsible for maintaining USP16 in the cytoplasm. We found that USP16 is only transiently retained in the nucleus following mitosis and then rapidly exported from this compartment. We also defined a non-canonical nuclear localization signal (NLS) sequence that plays a minimal role in directing USP16 into the nucleus. We further established that this DUB does not accumulate in the nucleus following DNA damage. Instead, only enforced nuclear localization of USP16 abolishes DNA double-strand break (DSB) repair, possibly due to unrestrained DUB activity. Thus, in contrast to the prevailing view, our data indicate that USP16 is actively excluded from the nucleus and that this DUB might indirectly regulate DSB repair.This article has an associated First Person interview with the first author of the paper.

3.
Oncogene ; 39(12): 2612-2623, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32020055

RESUMO

Neutrophils represent the immune system's first line of defense and are rapidly recruited into inflamed tissue. In cancer associated inflammation, phenotypic heterogeneity has been ascribed to this cell type, whereby neutrophils can manifest anti- or pro-metastatic functions depending on the cellular/micro-environmental context. Here, we demonstrate that pro-metastatic immature low-density neutrophils (iLDNs) more efficiently accumulate in the livers of mice bearing metastatic lesions compared with anti-metastatic mature high-density neutrophils (HDNs). Transcriptomic analyses reveal enrichment of a migration signature in iLDNs relative to HDNs. We find that conditioned media derived from liver-metastatic breast cancer cells, but not lung-metastatic variants, specifically induces chemotaxis of iLDNs and not HDNs. Chemotactic responses are due to increased surface expression of C3aR in iLDNs relative to HDNs. In addition, we detect elevated secretion of cancer-cell derived C3a from liver-metastatic versus lung-metastatic breast cancer cells. Perturbation of C3a/C3aR signaling axis with either a small molecule inhibitor, SB290157, or reducing the levels of secreted C3a from liver-metastatic breast cancer cells by short hairpin RNAs, can abrogate the chemotactic response of iLDNs both in vitro and in vivo, respectively. Together, these data reveal novel mechanisms through which iLDNs prefentially accumulate in liver tissue harboring metastases in response to tumor-derived C3a secreted from the liver-aggressive 4T1 breast cancer cells.

4.
Oncogene ; 39(14): 2996-3014, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32042114

RESUMO

The presence of an immature tumor vascular network contributes to cancer dissemination and the development of resistance to therapies. Strategies to normalize the tumor vasculature are therefore of significant therapeutic interest for cancer treatments. VEGF inhibitors are used clinically to normalize tumor blood vessels. However, the time frame and dosage of these inhibitors required to achieve normalization is rather narrow, and there is a need to identify additional signaling targets to attain vascular normalization. In addition to VEGF, the endothelial-specific receptor Alk1 plays a critical role in vascular development and promotes vascular remodeling and maturation. Therefore, we sought to evaluate the effects of the Alk1 ligand BMP9 on tumor vascular formation. BMP9 overexpression in Lewis Lung Carcinoma (LLC) tumors significantly delayed tumor growth. Blood vessels in BMP9-overexpressing LLC tumors displayed markers of vascular maturation and were characterized by increased perivascular cell coverage. Tumor vasculature normalization was associated with decreased permeability and increased perfusion. These changes in vascular function in BMP9-overexpressing LLC tumors resulted in significant alterations of the tumor microenvironment, characterized by a decrease in hypoxia and an increase in immune infiltration. In conclusion, we show that BMP9 promotes vascular normalization in LLC tumors that leads to changes in the microenvironment.

5.
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.

6.
Nat Genet ; 51(12): 1702-1713, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31768071

RESUMO

Childhood brain tumors have suspected prenatal origins. To identify vulnerable developmental states, we generated a single-cell transcriptome atlas of >65,000 cells from embryonal pons and forebrain, two major tumor locations. We derived signatures for 191 distinct cell populations and defined the regional cellular diversity and differentiation dynamics. Projection of bulk tumor transcriptomes onto this dataset shows that WNT medulloblastomas match the rhombic lip-derived mossy fiber neuronal lineage and embryonal tumors with multilayered rosettes fully recapitulate a neuronal lineage, while group 2a/b atypical teratoid/rhabdoid tumors may originate outside the neuroectoderm. Importantly, single-cell tumor profiles reveal highly defined cell hierarchies that mirror transcriptional programs of the corresponding normal lineages. Our findings identify impaired differentiation of specific neural progenitors as a common mechanism underlying these pediatric cancers and provide a rational framework for future modeling and therapeutic interventions.


Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Encéfalo/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Animais , Encéfalo/patologia , Linhagem Celular Tumoral , Humanos , Lactente , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/patologia , Fibras Nervosas/patologia , Fibras Nervosas/fisiologia , Prosencéfalo/citologia , Prosencéfalo/embriologia , Tumor Rabdoide/genética , Tumor Rabdoide/patologia , Análise de Célula Única
7.
Elife ; 82019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30969169

RESUMO

The ability to isolate rare live cells within a heterogeneous population based solely on visual criteria remains technically challenging, due largely to limitations imposed by existing sorting technologies. Here, we present a new method that permits labeling cells of interest by attaching streptavidin-coated magnetic beads to their membranes using the lasers of a confocal microscope. A simple magnet allows highly specific isolation of the labeled cells, which then remain viable and proliferate normally. As proof of principle, we tagged, isolated, and expanded individual cells based on three biologically relevant visual characteristics: i) presence of multiple nuclei, ii) accumulation of lipid vesicles, and iii) ability to resolve ionizing radiation-induced DNA damage foci. Our method constitutes a rapid, efficient, and cost-effective approach for isolation and subsequent characterization of rare cells based on observable traits such as movement, shape, or location, which in turn can generate novel mechanistic insights into important biological processes.


Assuntos
Separação Celular/métodos , Campos Magnéticos , Coloração e Rotulagem/métodos , Estreptavidina/metabolismo , Animais , Linhagem Celular , Humanos
8.
SLAS Technol ; 24(3): 298-307, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30707854

RESUMO

Multiplexing strategies, which greatly increase the number of simultaneously measured parameters in single experiments, are now being widely implemented by both the pharmaceutical industry and academic researchers. Color has long been used to identify biological signals and, when combined with molecular barcodes, has substantially enhanced the depth of multiplexed sample characterization. Moreover, the recent advent of DNA barcodes has led to an explosion of innovative cell sequencing approaches. Novel barcoding strategies also show great promise for encoding spatial information in transcriptomic studies, and for precise assessment of molecular abundance. Both color- and DNA-based barcodes can be conveniently analyzed with either a microscope or a cytometer, or via DNA sequencing. Here we review the basic principles of several technologies used to create barcodes and detail the type of samples that can be identified with such tags.


Assuntos
Técnicas Citológicas/métodos , Técnicas de Sonda Molecular , Coloração e Rotulagem/métodos , Automação Laboratorial/métodos , Citometria de Fluxo , Ensaios de Triagem em Larga Escala/métodos , Microscopia
9.
Nat Commun ; 10(1): 22, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30604754

RESUMO

Mechanisms regulating B cell development, activation, education in the germinal center (GC) and differentiation, underpin the humoral immune response. Protein arginine methyltransferase 5 (Prmt5), which catalyzes most symmetric dimethyl arginine protein modifications, is overexpressed in B cell lymphomas but its function in normal B cells is poorly defined. Here we show that Prmt5 is necessary for antibody responses and has essential but distinct functions in all proliferative B cell stages in mice. Prmt5 is necessary for B cell development by preventing p53-dependent and p53-independent blocks in Pro-B and Pre-B cells, respectively. By contrast, Prmt5 protects, via p53-independent pathways, mature B cells from apoptosis during activation, promotes GC expansion, and counters plasma cell differentiation. Phenotypic and RNA-seq data indicate that Prmt5 regulates GC light zone B cell fate by regulating transcriptional programs, achieved in part by ensuring RNA splicing fidelity. Our results establish Prmt5 as an essential regulator of B cell biology.


Assuntos
Linfócitos B/fisiologia , Proliferação de Células/fisiologia , Centro Germinativo/fisiologia , Imunidade Humoral/fisiologia , Proteína-Arginina N-Metiltransferases/fisiologia , Animais , Apoptose/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Diferenciação Celular/imunologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Centro Germinativo/citologia , Humanos , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Cultura Primária de Células , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Transdução de Sinais/fisiologia , Trichostrongyloidea/imunologia , Tricostrongiloidíase/imunologia , Tricostrongiloidíase/parasitologia , Proteína Supressora de Tumor p53/metabolismo
10.
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
11.
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.

12.
Cancer Res ; 78(19): 5561-5573, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30072396

RESUMO

Intrinsic and acquired resistance to cisplatin remains a primary hurdle to treatment of high-grade serous ovarian cancer (HGSOC). Cisplatin selectively kills tumor cells by inducing DNA crosslinks that block replicative DNA polymerases. Single-stranded DNA (ssDNA) generated at resulting stalled replication forks (RF) is bound and protected by heterotrimeric replication protein A (RPA), which then serves as a platform for recruitment and activation of replication stress response factors. Cells deficient in this response are characterized by extensive ssDNA formation and excessive RPA recruitment that exhausts the available pool of RPA, which (i) inhibits RPA-dependent processes such as nucleotide excision repair (NER) and (ii) causes catastrophic failure of blocked RF. Here, we investigated the influence of RPA availability on chemosensitivity using a panel of human HGSOC cell lines. Our data revealed a striking correlation among these cell lines between cisplatin sensitivity and the inability to efficiently repair DNA via NER, specifically during S phase. Such defects in NER were attributable to RPA exhaustion arising from aberrant activation of DNA replication origins during replication stress. Reduced RPA availability promoted Mre11-dependent degradation of nascent DNA at stalled RF in cell lines exhibiting elevated sensitivity to cisplatin. Strikingly, defective S-phase NER, RF instability, and cisplatin sensitivity could all be rescued by ectopic overexpression of RPA. Taken together, our findings indicate that RPA exhaustion represents a major determinant of cisplatin sensitivity in HGSOC cell lines.Significance: The influence of replication protein A exhaustion on cisplatin sensitivity harbors important implications toward improving therapy of various cancers that initially respond to platinum-based agents but later relapse due to intrinsic or acquired drug resistance. Cancer Res; 78(19); 5561-73. ©2018 AACR.


Assuntos
Cisplatino/farmacologia , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Proteína de Replicação A/metabolismo , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , DNA de Cadeia Simples/genética , Feminino , Humanos , RNA Interferente Pequeno/metabolismo
13.
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
14.
Curr Eye Res ; 42(12): 1620-1627, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28937876

RESUMO

PURPOSE: To perform a pilot study of the neuro-peripapillary retinal tissue deformation during the cardiac cycle among healthy eyes, ocular hypertensive (OHT), open angle glaucoma suspect (OAG-S), and early open angle glaucoma (EOAG) patients using video rate optical coherence tomography (OCT) image series. METHODS: OCT line scan sequences of the same region of the optic nerve head (ONH) were obtained from 15 EOAG, 6 OHT, 10 OAG-S, and 10 healthy age-matched eyes. One eye per patient was studied. Changes in the axial distance between the inferotemporal peripapillary retina and the prelaminar tissue, in time, were determined using an automated custom made algorithm. Linear correlations between this neuro-peripapillary retinal (N-PP) deformation and variables measured during the full ophthalmic examination are analyzed. RESULTS: Healthy eyes showed larger N-PP deformation (4.8 ± 1 µm) than the OHT (3.5 ± 0.3 µm, p = 0.015), OAG-S (3.8 ± 0.8 µm, p = 0.045), and EOAG (3.2 ± 0.7 µm, p < 0.001) groups. Eyes with lower ocular pulse amplitude, thinner RNFL's, or worse visual fields showed smaller N-PP deformation, depending on the diagnosis. A linear model to explain deformation within the EOAG group with intraocular pressure and systolic perfusion pressure as predictors was found to be significant (R2 = 0.767, p < 0.001). CONCLUSIONS: Smaller mean N-PP deformation was observed in the EOAG, OAG-S, and OHT groups compared to healthy eyes in this pilot study. The measured deformation correlated with risk factors for the glaucomatous optic neuropathy, but these correlations varied depending on the diagnosis. The role of pulsatile neuro-peripapillary retinal deformation in the pathophysiology of OAG remains to be determined.


Assuntos
Corioide/irrigação sanguínea , Glaucoma de Ângulo Aberto/fisiopatologia , Pressão Intraocular/fisiologia , Disco Óptico/fisiopatologia , Fluxo Pulsátil/fisiologia , Retina/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Fenômenos Biomecânicos , Feminino , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Fibras Nervosas , Hipertensão Ocular/fisiopatologia , Disco Óptico/diagnóstico por imagem , Projetos Piloto , Retina/diagnóstico por imagem , Células Ganglionares da Retina , Fatores de Risco , Tomografia de Coerência Óptica , Campos Visuais
15.
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
16.
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
17.
J Neurosci ; 37(14): 3824-3839, 2017 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-28275163

RESUMO

Axonal degeneration is a pathophysiological mechanism common to several neurodegenerative diseases. The slow Wallerian degeneration (WldS) mutation, which results in reduced axonal degeneration in the central and peripheral nervous systems, has provided insight into a redox-dependent mechanism by which axons undergo self-destruction. We studied early molecular events in axonal degeneration with single-axon laser axotomy and time-lapse imaging, monitoring the initial changes in transected axons of purified retinal ganglion cells (RGCs) from wild-type and WldS rat retinas using a polarity-sensitive annexin-based biosensor (annexin B12-Cys101,Cys260-N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylenediamine). Transected axons demonstrated a rapid and progressive change in membrane phospholipid polarity, manifested as phosphatidylserine externalization, which was significantly delayed and propagated more slowly in axotomized WldS RGCs compared with wild-type axons. Delivery of bis(3-propionic acid methyl ester)phenylphosphine borane complex, a cell-permeable intracellular disulfide-reducing drug, slowed the onset and velocity of phosphatidylserine externalization in wild-type axons significantly, replicating the WldS phenotype, whereas extracellular redox modulation reversed the WldS phenotype. These findings are consistent with an intra-axonal redox mechanism for axonal degeneration associated with the initiation and propagation of phosphatidylserine externalization after axotomy.SIGNIFICANCE STATEMENT Axonal degeneration is a neuronal process independent of somal apoptosis, the propagation of which is unclear. We combined single-cell laser axotomy with time-lapse imaging to study the dynamics of phosphatidylserine externalization immediately after axonal injury in purified retinal ganglion cells. The extension of phosphatidylserine externalization was slowed and delayed in Wallerian degeneration slow (WldS) axons and this phenotype could be reproduced by intra-axonal disulfide reduction in wild-type axons and reversed by extra-axonal reduction in WldS axons. These results are consistent with a redox mechanism for propagation of membrane polarity asymmetry in axonal degeneration.


Assuntos
Membrana Celular/metabolismo , Polaridade Celular/fisiologia , Células Ganglionares da Retina/metabolismo , Degeneração Walleriana/metabolismo , Animais , Animais Recém-Nascidos , Axotomia/efeitos adversos , Membrana Celular/patologia , Células Cultivadas , Feminino , Masculino , Oxirredução , Gravidez , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Células Ganglionares da Retina/patologia , Degeneração Walleriana/patologia
18.
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
19.
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
20.
Am J Pathol ; 186(12): 3100-3116, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27768863

RESUMO

Retinopathy of prematurity (ROP), the most common cause of blindness in premature infants, has long been associated with inner retinal alterations. However, recent studies reveal outer retinal dysfunctions in patients formerly afflicted with ROP. We have recently demonstrated that choroidal involution occurs early in retinopathy. Herein, we investigated the mechanisms underlying the choroidal involution and its long-term impact on retinal function. An oxygen-induced retinopathy (OIR) model was used. In vitro and ex vivo assays were applied to evaluate cytotoxic effects of IL-1ß on choroidal endothelium. Electroretinogram was used to evaluate visual function. We found that proinflammatory IL-1ß was markedly increased in retinal pigment epithelium (RPE)/choroid and positively correlated with choroidal degeneration in the early stages of retinopathy. IL-1ß was found to be cytotoxic to choroid in vitro, ex vivo, and in vivo. Long-term effects on choroidal involution included a hypoxic outer neuroretina, associated with a progressive loss of RPE and photoreceptors, and visual deterioration. Early inhibition of IL-1ß receptor preserved choroid, decreased subretinal hypoxia, and prevented RPE/photoreceptor death, resulting in life-long improved visual function in IL-1 receptor antagonist-treated OIR animals. Together, these findings suggest a critical role for IL-1ß-induced choroidal degeneration in outer retinal dysfunction. Neonatal therapy using IL-1 receptor antagonist preserves choroid and prevents protracted outer neuroretinal anomalies in OIR, suggesting IL-1ß as a potential therapeutic target in ROP.


Assuntos
Doenças da Coroide/fisiopatologia , Interleucina-1beta/metabolismo , Retinopatia da Prematuridade/fisiopatologia , Animais , Animais Recém-Nascidos , Corioide/metabolismo , Corioide/fisiopatologia , Doenças da Coroide/etiologia , Doenças da Coroide/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Eletrorretinografia , Endotélio/metabolismo , Humanos , Recém-Nascido , Oxigênio/efeitos adversos , Células Fotorreceptoras/metabolismo , Ratos , Ratos Sprague-Dawley , Retina/metabolismo , Retina/fisiopatologia , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/fisiopatologia , Retinopatia da Prematuridade/induzido quimicamente , Retinopatia da Prematuridade/etiologia , Retinopatia da Prematuridade/metabolismo
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