Dataset Information

Systemic treatment with cigarette smoke extract affects zebrafish visual behaviour, intraocular vasculature morphology and outer segment phagocytosis.

ABSTRACT:

Introduction

Cigarette smoking adversely affects multiple aspects of human health including eye disorders such as age-related macular degeneration, cataracts and dry eye disease. However, there remains a knowledge gap in how constituents of cigarette smoke affect vision and retinal biology. We used zebrafish to assess effects of short-term acute exposure to cigarette smoke extract (CSE) on visual behaviour and retinal biology.

Methods

Zebrafish larvae with a developed visual system at three days post-fertilization (dpf) were exposed to CSE for 4, 24 or 48 hours. Visual behaviour, hyaloid vasculature morphology, retinal histology, oxidative stress gene expression and outer segment phagocytosis were investigated using visual behavioural optokinetic and visual motor response assays (OKR and VMR), microscopy (light, fluorescence and transmission electron microscopy), and real-time PCR.

Results

In zebrafish larvae, 48 hours of CSE treatment resulted in significantly reduced visual behaviour. Larvae treated with 10, 15 or 20 μg/mL CSE showed an average of 13.7, 10.7 or 9.4 saccades per minute, respectively, significantly lower compared with 0.05% DMSO controls (p=0.0093, p=0.0004 and p<0.0001, respectively) that exhibited 19.7 saccades per minute. The diameter of intraocular vessels increased from 4.833 μm in 0.05% DMSO controls to 5.885 μm in the 20 μg/mL CSE-treated larvae (p=0.0333). Biometry analysis highlighted a significant axial length elongation in 20 μg/mL CSE-treated larvae (216.9 μm, p<0.0001) compared to 0.05% dimethyl sulfoxide (DMSO) controls (205.1 μm). Larvae exposed to 20 μg/mL CSE had significantly (p=0.0002) higher numbers of RPE phagosomes compared to vehicle controls (0.1425 and 0.093 phagosomes/μm RPE, respectively).

Conclusions

Zebrafish larvae with a developed visual system display apparent defects in visual behaviour and retinal biology after acute exposure to CSE, establishing a valuable in vivo model to investigate ocular disorders related to cigarette smoke.

SUBMITTER: Gomez Sanchez A

PROVIDER: S-EPMC10822043 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Publications

Systemic treatment with cigarette smoke extract affects zebrafish visual behaviour, intraocular vasculature morphology and outer segment phagocytosis.

Gómez Sánchez Alicia A Colucci Patrizia P Moran Ailis A Moya López Alexandro A Colligris Basilio B Álvarez Yolanda Y Kennedy Breandán N BN

Open research Europe 20231123

<h4>Introduction</h4>Cigarette smoking adversely affects multiple aspects of human health including eye disorders such as age-related macular degeneration, cataracts and dry eye disease. However, there remains a knowledge gap in how constituents of cigarette smoke affect vision and retinal biology. We used zebrafish to assess effects of short-term acute exposure to cigarette smoke extract (CSE) on visual behaviour and retinal biology.<h4>Methods</h4>Zebrafish larvae with a developed visual syste ...[more]

PMID: 38283058

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Project description:Introduction/rationaleIn chronic obstructive pulmonary disease (COPD), defective macrophage phagocytic clearance of cells undergoing apoptosis by efferocytosis may lead to secondary necrosis of the uncleared cells and contribute to airway inflammation. The precise mechanisms for this phenomenon remain unknown. LC3-associated phagocytosis (LAP) is indispensable for effective efferocytosis. We hypothesized that cigarette smoke inhibits the regulators of LAP pathway, potentially contributing to the chronic airways inflammation associated with COPD.MethodsBronchoalveolar (BAL)-derived alveolar macrophages, lung tissue macrophages obtained from lung resection surgery, and monocyte-derived macrophages (MDM) were prepared from COPD patients and control participants. Lung/airway samples from mice chronically exposed to cigarette smoke were also investigated. Differentiated THP-1 cells were exposed to cigarette smoke extract (CSE). The LAP pathway including Rubicon, as an essential regulator of LAP, efferocytosis and inflammation was examined using western blot, ELISA, flow cytometry, and/or immunofluorescence.ResultsRubicon was significantly depleted in COPD alveolar macrophages compared with non-COPD control macrophages. Rubicon protein in alveolar macrophages of cigarette smoke-exposed mice and cigarette smoke-exposed MDM and THP-1 was decreased with a concomitant impairment of efferocytosis. We also noted increased expression of LC3 which is critical for LAP pathway in COPD and THP-1 macrophages. Furthermore, THP-1 macrophages exposed to cigarette smoke extract exhibited higher levels of other key components of LAP pathway including Atg5 and TIM-4. There was a strong positive correlation between Rubicon protein expression and efferocytosis.ConclusionLAP is a requisite for effective efferocytosis and an appropriate inflammatory response, which is impaired by Rubicon deficiency. Our findings suggest dysregulated LAP due to reduced Rubicon as a result of CSE exposure. This phenomenon could lead to a failure of macrophages to effectively process phagosomes containing apoptotic cells during efferocytosis. Restoring Rubicon protein expression has unrecognized therapeutic potential in the context of disease-related modifications caused by exposure to cigarette smoke.

Project description:BackgroundThe retinal rod outer segment is a sensory cilium that is specialized for the conversion of light into an electrical signal. Within the cilium, up to several thousand membranous disks contain as many as a billion copies of rhodopsin for efficient photon capture. Disks are continually turned over, requiring the daily synthesis of a prodigious amount of rhodopsin. To promote axial diffusion in the aqueous cytoplasm, the disks have one or more incisures. Across vertebrates, the range of disk diameters spans an order of magnitude, and the number and length of the incisures vary considerably, but the mechanisms controlling disk architecture are not well understood. The finding that transgenic mice overexpressing rhodopsin have enlarged disks lacking an incisure prompted us to test whether lowered rhodopsin levels constrain disk assembly.Methodology/principal findingsThe structure and function of rods from hemizygous rhodopsin knockout (R+/-) mice with decreased rhodopsin expression were analyzed by transmission electron microscopy and single cell recording. R+/- rods were structurally altered in three ways: disk shape changed from circular to elliptical, disk surface area decreased, and the single incisure lengthened to divide the disk into two sections. Photocurrent responses to flashes recovered more rapidly than normal. A spatially resolved model of phototransduction indicated that changes in the packing densities of rhodopsin and other transduction proteins were responsible. The decrease in aqueous outer segment volume and the lengthened incisure had only minor effects on photon response amplitude and kinetics.Conclusions/significanceRhodopsin availability limits disk assembly and outer segment girth in normal rods. The incisure may buffer the supply of structural proteins needed to form larger disks. Decreased rhodopsin level accelerated photoresponse kinetics by increasing the rates of molecular collisions on the membrane. Faster responses, together with fewer rhodopsins, combine to lower overall sensitivity of R+/- rods to light.

Dataset Information

Systemic treatment with cigarette smoke extract affects zebrafish visual behaviour, intraocular vasculature morphology and outer segment phagocytosis.

Introduction

Methods

Results

Conclusions

Publications

Systemic treatment with cigarette smoke extract affects zebrafish visual behaviour, intraocular vasculature morphology and outer segment phagocytosis.

Similar Datasets

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