Preparing a Single Cell Suspension from Zebrafish Retinal Tissue for Flow Cytometric Cell Sorting of Müller Glia

Allan, Kristin; DiCicco, Rose M; Ramos, Michael; Asosingh, Kewal; Yuan, Alex

doi:10.1002/cyto.a.23936

Cited by 6 publications

(4 citation statements)

References 58 publications

(91 reference statements)

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“…Analyses of the cell cycle distribution of neurons and neuronal precursor cells were performed as previously reported [ 47 , 84 ]. Briefly, Tg(elavl3:EGFP) zebrafish larvae (6 dpf, 100 larvae per sample) which had been digested and homogenized, were washed twice with ice-cold PBS, and then were fixed in 70% ethanol at 4 °C for overnight.…”

Section: Methodsmentioning

confidence: 99%

Wnt/β-Catenin Signaling Pathway Is Strongly Implicated in Cadmium-Induced Developmental Neurotoxicity and Neuroinflammation: Clues from Zebrafish Neurobehavior and In Vivo Neuroimaging

Liu

Tian

et al. 2022

IJMS

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Cadmium (Cd) is a toxic heavy metal and worldwide environmental pollutant which seriously threatens human health and ecosystems. It is easy to be adsorbed and deposited in organisms, exerting adverse effects on various organs including the brain. In a very recent study, making full use of a zebrafish model in both high-throughput behavioral tracking and live neuroimaging, we explored the potential developmental neurotoxicity of Cd2+ at environmentally relevant levels and identified multiple connections between Cd2+ exposure and neurodevelopmental disorders as well as microglia-mediated neuroinflammation, whereas the underlying neurotoxic mechanisms remained unclear. The canonical Wnt/β-catenin signaling pathway plays crucial roles in many biological processes including neurodevelopment, cell survival, and cell cycle regulation, as well as microglial activation, thereby potentially presenting one of the key targets of Cd2+ neurotoxicity. Therefore, in this follow-up study, we investigated the implication of the Wnt/β-catenin signaling pathway in Cd2+-induced developmental disorders and neuroinflammation and revealed that environmental Cd2+ exposure significantly affected the expression of key factors in the zebrafish Wnt/β-catenin signaling pathway. In addition, pharmacological intervention of this pathway via TWS119, which can increase the protein level of β-catenin and act as a classical activator of the Wnt signaling pathway, could significantly repress the Cd2+-induced cell cycle arrest and apoptosis, thereby attenuating the inhibitory effects of Cd2+ on the early development, behavior, and activity, as well as neurodevelopment of zebrafish larvae to a certain degree. Furthermore, activation and proliferation of microglia, as well as the altered expression profiles of genes associated with neuroimmune homeostasis triggered by Cd2+ exposure could also be significantly alleviated by the activation of the Wnt/β-catenin signaling pathway. Thus, this study provided novel insights into the cellular and molecular mechanisms of Cd2+ toxicity on the vertebrate central nervous system (CNS), which might be helpful in developing pharmacotherapies to mitigate the neurological disorders resulting from exposure to Cd2+ and many other environmental heavy metals.

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Section: Methodsmentioning

confidence: 99%

Wnt/β-Catenin Signaling Pathway Is Strongly Implicated in Cadmium-Induced Developmental Neurotoxicity and Neuroinflammation: Clues from Zebrafish Neurobehavior and In Vivo Neuroimaging

Liu

Tian

et al. 2022

IJMS

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“…RECs were isolated from the retinae in four experimental groups using the flow sorting method as described before [ 44 , 45 ]. In each experimental group, mice were euthanized, and their retinas were carefully enucleated for subsequent analysis.…”

Section: Methodsmentioning

confidence: 99%

“…Isolation of CD31 + RECs Using Flow Sorting. RECs were isolated from the retinae in four experimental groups using the flow sorting method as described before [44,45].…”

Section: Cellular Fractionationmentioning

confidence: 99%

lncRNA ZFAS1 Positively Facilitates Endothelial Ferroptosis via miR-7-5p/ACSL4 Axis in Diabetic Retinopathy

Liu

Zhang

Yang

et al. 2022

Oxidative Medicine and Cellular Longevity

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Accumulating evidence has suggested the significant role of long noncoding RNAs (lncRNA) in regulating ferroptosis, while its regulatory mechanism in diabetic retinopathy (DR) remains unelucidated. In this work, we first demonstrated that lncRNA zinc finger antisense 1 (ZFAS1) is upregulated in high glucose-cultured human retinal endothelial cells (hRECs) and ZFAS1 inhibition attenuated high glucose- (HG-) induced ferroptosis, which was evidenced by cell viability, total iron and ferrous iron levels, reactive oxygen species (ROS) level, and Glutathione Peroxidase 4 (GPX4) expression detection. Mechanistically, we validated that ZFAS1 may act as a competing endogenous RNA by competitively binding with microRNA-7-5p (miR-7-5p) and modulating the expression of its downstream molecule acyl-CoA synthetase long-chain family member 4 (ACSL4), which is now identified as a classic driver gene of ferroptosis process. In conclusion, our results demonstrate that HG-induced ZFAS1 elevation activates ferroptosis in hRECs and the ZFAS1/miR-7-5p/ACSL4 axis may serve as a therapeutic target for endothelial dysfunction in DR.

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“…AB, Tg(apoe:GFP) [78], Tg(coro1a:DsRed) [37], Tg(coro1a:eGFP), Tg(coro1a:Dendra2-NTR), Tg(coro1a:Kaede), Tg(HuC:GFP) [32], Tg(mpeg1:eGFP) [36] and Tg(krt4:Sce.Abp140-Venus) cy22 [79] strains were maintained according to the guidelines of experimental animal welfare from the Ministry of Science and Technology of the People's Republic of China (2006). All animal experiments were performed under approval from the Institutional Review Board of Southwest University (Chongqing, China).…”

Section: Zebrafishmentioning

confidence: 99%

Macrophages Rapidly Seal off the Punctured Zebrafish Larval Brain through a Vital Honeycomb Network Structure

Zou

Qin

et al. 2022

IJMS

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There is accumulating evidence that macrophages play additional important roles in tissue damage besides their typical phagocytosis. Although the aggregation of macrophages on injured sites has long been observed, few researchers have focused on the role of the overall structure of macrophage aggregation. In this study, we developed a standardized traumatic brain injury (TBI) model in zebrafish larvae to mimic edema and brain tissue spillage symptoms after severe brain trauma. Using time-lapse imaging, we showed that macrophages/microglia in zebrafish larvae responded rapidly and dominated the surface of injured tissue, forming a meaningful honeycomb network structure through their compact aggregation and connection. Disrupting this structure led to fatal edema-like symptoms with severe loss of brain tissue. Using the RNA-Seq, together with the manipulation of in vitro cell lines, we found that collagen IV was indispensable to the formation of honeycomb network structures. Our study thus revealed a novel perspective regarding macrophages forming a protective compact structure with collagen IV. This honeycomb network structure acted as a physical barrier to prevent tissue loss and maintain brain homeostasis after TBI. This study may provide new evidence of macrophages’ function for the rapid protection of brain tissue after brain injury.

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Preparing a Single Cell Suspension from Zebrafish Retinal Tissue for Flow Cytometric Cell Sorting of Müller Glia

Cited by 6 publications

References 58 publications

Wnt/β-Catenin Signaling Pathway Is Strongly Implicated in Cadmium-Induced Developmental Neurotoxicity and Neuroinflammation: Clues from Zebrafish Neurobehavior and In Vivo Neuroimaging

Wnt/β-Catenin Signaling Pathway Is Strongly Implicated in Cadmium-Induced Developmental Neurotoxicity and Neuroinflammation: Clues from Zebrafish Neurobehavior and In Vivo Neuroimaging

lncRNA ZFAS1 Positively Facilitates Endothelial Ferroptosis via miR-7-5p/ACSL4 Axis in Diabetic Retinopathy

Macrophages Rapidly Seal off the Punctured Zebrafish Larval Brain through a Vital Honeycomb Network Structure

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