A Confocal Reflection Super‐Resolution Technique to Image Golgi‐Cox Stained Neurons

Sivaguru, Mayandi; Khaw, Yee Ming; Inoue, Makoto

doi:10.1111/jmi.12821

Cited by 15 publications

(19 citation statements)

References 67 publications

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“…Samples of Golgi-Cox-stained spinal cords were imaged using a Nikon A1 confocal scanning microscope under the confocal modality and CRSR modality (with minimized pinhole at 0.3 AU) using 20×/0.8 NA objective and 100×/1.49 NA oil objective, respectively [33]. A 405nm continuous wave laser was used, and the reflectance mirror (BS 20/80) was applied.…”

Section: Confocal Reflection Super-resolution (Crsr) Acquisitionmentioning

confidence: 99%

“…Four to eight z-stacks of spinal cord ventral roots from four to eight individual 50-μm-thick spinal cord sections per animal were visualized. A total of 220 dendrites (in 3-4 animals per condition) were included in our analyses (naïve, 60 dendrites; control EAE, 80 dendrites; Cxcr2 cKO EAE, 80 dendrites) using the filament tracer autopath function (Imaris), as previously described [33,34]. Importantly, Gaussian filter and background subtraction were applied to z-stacks of cropped individual dendrites prior to tracing filaments.…”

Section: Confocal Reflection Super-resolution (Crsr) Acquisitionmentioning

confidence: 99%

“…2a). Using our recently developed confocal reflection super-resolution (CRSR) technique [33], Student's t test revealed that control EAE mice exhibited significantly larger (*p < 0.05) neuronal somas than Cxcr2 cKO EAE mice during late phase disease ( Fig. 2b-d).…”

Section: Cxcr2 + Neutrophils Mediate Spinal Cord Neuronal Abnormalitimentioning

confidence: 99%

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Neutrophil-selective deletion of Cxcr2 protects against CNS neurodegeneration in a mouse model of multiple sclerosis

Khaw

Cunningham

Tierney

et al. 2020

J Neuroinflammation

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Background: Multiple sclerosis (MS) is a chronic debilitating immune-mediated disease of the central nervous system (CNS) driven by demyelination and gray matter neurodegeneration. We previously reported an experimental autoimmune encephalomyelitis (EAE) MS mouse model with elevated serum CXCL1 that developed severe and prolonged neuron damage. Our findings suggested that CXCR2 signaling may be important in neuronal damage, thus implicating neutrophils, which express CXCR2 in abundance, as a potential cell type involved. The goals of this study were to determine if CXCR2 signaling in neutrophils mediate neuronal damage and to identify potential mechanisms of damage. Methods: EAE was induced in wild-type control and neutrophil-specific Cxcr2 knockout (Cxcr2 cKO) mice by repeated high-dose injections of heat-killed Mycobacterium tuberculosis and MOG 35-55 peptide. Mice were examined daily for motor deficit. Serum CXCL1 level was determined at different time points throughout disease development. Neuronal morphology in Golgi-Cox stained lumbar spinal cord ventral horn was assessed using recently developed confocal reflection super-resolution technique. Immune cells from CNS and lymphoid organs were quantified by flow cytometry. CNS-derived neutrophils were co-cultured with neuronal crest cells and neuronal cell death was measured. Neutrophils isolated from lymphoid organs were examined for expression of reactive oxygen species (ROS) and ROS-related genes. Thioglycolate-activated neutrophils were isolated, treated with recombinant CXCL1, and measured for ROS production. Results: Cxcr2 cKO mice had less severe disease symptoms at peak and late phase when compared to control mice with similar levels of CNS-infiltrating neutrophils and other immune cells despite high levels of circulating CXCL1. Additionally, Cxcr2 cKO mice had significantly reduced CNS neuronal damage in the ventral horn of the spinal cord. Neutrophils isolated from control EAE mice induced vast neuronal cell death in vitro when compared with neutrophils isolated from Cxcr2 cKO EAE mice. Neutrophils isolated from control EAE mice, but not Cxcr2 cKO mice, exhibited elevated ROS generation, in addition to heightened Ncf1 and Il1b transcription. Furthermore, recombinant CXCL1 was sufficient to significantly increase neutrophils ROS production. Conclusions: CXCR2 signal in neutrophils is critical in triggering CNS neuronal damage via ROS generation, which leads to prolonged EAE disease. These findings emphasize that CXCR2 signaling in neutrophils may be a viable target for therapeutic intervention against CNS neuronal damage.

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Section: Confocal Reflection Super-resolution (Crsr) Acquisitionmentioning

confidence: 99%

Section: Confocal Reflection Super-resolution (Crsr) Acquisitionmentioning

confidence: 99%

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Neutrophil-selective deletion of Cxcr2 protects against CNS neurodegeneration in a mouse model of multiple sclerosis

Khaw

Cunningham

Tierney

et al. 2020

J Neuroinflammation

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“…3a ). We then sought to determine dendritic spine density using the recently reported Golgi-visualizing confocal reflection super-resolution method (CRSR) 24 . Reduced spine density was found in the dendrites of neurons that reside in the lumbar spinal cord ventral horn in control EAE mice and ELT-EAE mice at 30 dpi, compared to naïve and ELT mice without EAE (Fig.…”

Section: Resultsmentioning

confidence: 99%

Early-life-trauma triggers interferon-β resistance and neurodegeneration in a multiple sclerosis model via downregulated β1-adrenergic signaling

Khaw

Majid

et al. 2021

Nat Commun

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Environmental triggers have important functions in multiple sclerosis (MS) susceptibility, phenotype, and trajectory. Exposure to early life trauma (ELT) has been associated with higher relapse rates in MS patients; however, the underlying mechanisms are not well-defined. Here we show ELT induces mechanistic and phenotypical alterations during experimental autoimmune encephalitis (EAE). ELT sustains downregulation of immune cell adrenergic receptors, which can be attributed to chronic norepinephrine circulation. ELT-subjected mice exhibit interferon-β resistance and neurodegeneration driven by lymphotoxin and CXCR2 involvement. These phenotypic changes are observed in control EAE mice treated with β1 adrenergic receptor antagonist. Conversely, β1 adrenergic receptor agonist treatment to ELT mice abrogates phenotype changes via restoration of immune cell β1 adrenergic receptor function. Our results indicate that ELT alters EAE phenotype via downregulation of β1 adrenergic signaling in immune cells. These results have implications for the effect of environmental factors in provoking disease heterogeneity and might enable prediction of long-term outcomes in MS.

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“…Most modifications have aimed to decrease the time required for the procedure while maintaining the quality of the staining. However, the modified protocols reported are still time-consuming with insufficient clarity of the neuronal morphology (Du, 2019 ; Sivaguru et al, 2019 ). Aside from this, the long fixation period turns the tissue brittle, making sectioning more difficult.…”

Section: Introductionmentioning

confidence: 99%

Optimized Golgi-Cox Staining Validated in the Hippocampus of Spared Nerve Injury Mouse Model

et al. 2020

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A Confocal Reflection Super‐Resolution Technique to Image Golgi‐Cox Stained Neurons

Cited by 15 publications

References 67 publications

Neutrophil-selective deletion of Cxcr2 protects against CNS neurodegeneration in a mouse model of multiple sclerosis

Neutrophil-selective deletion of Cxcr2 protects against CNS neurodegeneration in a mouse model of multiple sclerosis

Early-life-trauma triggers interferon-β resistance and neurodegeneration in a multiple sclerosis model via downregulated β1-adrenergic signaling

Optimized Golgi-Cox Staining Validated in the Hippocampus of Spared Nerve Injury Mouse Model

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