Mechanical spinal cord transection in larval zebrafish and subsequent whole-mount histological processing

John, Nora; Kolb, Julia; Wehner, Daniel

doi:10.1016/j.xpro.2021.101093

Cited by 8 publications

(8 citation statements)

References 18 publications

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“…A detailed protocol for inducing spinal cord lesions in zebrafish larvae has been previously described ( 55 ). Briefly, zebrafish larvae (3 dpf) were anesthetized in E3 medium containing 0.02% MS-222.…”

Section: Methodsmentioning

confidence: 99%

“…A detailed protocol for ISH on whole-mount zebrafish larvae with digoxygenin (DIG)-labeled antisense probes has been described in ( 55 ). In brief, terminally anesthetized larvae were fixed in 4% PFA (Thermo Fisher Scientific Cat#28908) in PBS and treated with Proteinase K (Invitrogen Cat#25530-049) followed by re-fixation for 15 min in 4% PFA in PBS.…”

Section: Methodsmentioning

confidence: 99%

“…A detailed protocol for IF on whole-mount zebrafish larvae has been previously described ( 55 ). Briefly, terminally anesthetized larvae were fixed in 4% PFA (Thermo Fisher Scientific Cat#28908) in PBS for 1 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

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Small leucine-rich proteoglycans inhibit CNS regeneration by modifying the structural and mechanical properties of the lesion environment

Kolb

John

et al. 2022

Preprint

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Extracellular matrix (ECM) deposition after central nervous system (CNS) injury leads to inhibitory scarring in mammals, whereas it facilitates axon regeneration in the zebrafish. However, the molecular basis of these different fates is not understood. Here, we identify small leucine-rich proteoglycans (SLRPs) as a causal factor in regeneration failure. We demonstrate that the SLRPs Chondroadherin, Fibromodulin, Lumican, and Prolargin are enriched in human, but not zebrafish, CNS lesions. Targeting SLRPs to the zebrafish injury ECM inhibits axon regeneration and functional recovery. Mechanistically, we find that SLRPs confer structural and mechanical properties to the lesion environment that are adverse to axon growth. Our study reveals SLRPs as previously unknown inhibitory ECM factors in the human CNS that impair axon regeneration by modifying tissue mechanics and structure.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Small leucine-rich proteoglycans inhibit CNS regeneration by modifying the structural and mechanical properties of the lesion environment

Kolb

John

et al. 2022

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“…For example, transgenic lines can be used to look at individual larvae such that a specific injury can be monitored from time of injury to regeneration. Most researchers, including ourselves, use the cloaca as a marker for spatial reproducibility (Briona and Dorsky, 2014b;John et al, 2022); however, manual transection can lead to slight differences in extent of injury including: partial transections, size of injury, amount of secondary injury, and extraneous tissue damage (including the notochord).…”

Section: Discussionmentioning

confidence: 99%

Optimizing spinal cord injury in zebrafish larvae: effects of age on the injury response

Underwood,

Walker,

Garrett

et al. 2023

Preprint

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Zebrafish are an increasingly popular model to study spinal cord injury (SCI) regeneration. The transparency of larval zebrafish makes them ideal to study cellular processes in real time. Standardized approaches, including age of injury, are not readily available making comparisons of the results with other models challenging. In this study, we systematically examined the response to spinal cord transection of larval zebrafish at three different ages (3-7 days post fertilization or dpf) to determine whether the developmental complexity of the central nervous system affects the overall response to SCI. We then used imaging and behavioral analysis to evaluate whether differences existed based on the age of injury. All ages of larval zebrafish upregulated the required genes for glial bridge formation, ctgfa and gfap, at the site of injury, consistent with studies from adult zebrafish. Though all larval ages upregulated factors required to promote glial bridging, young larval zebrafish (3 dpf) were better able to regenerate axons independent of the glial bridge, unlike older zebrafish (7 dpf). Consistent with this data, locomotor experiments demonstrated that some swimming behavior occurs independent of glial bridge formation, further highlighting the need for standardization of this model and recovery assays. Overall, we found subtle cellular differences based on the age of transection in zebrafish, underlining the importance of considering age when designing experiments aimed at understanding regeneration.

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“…Whole-mount TUNEL labelling: Terminally anesthetized larvae were fixed in 4% PFA (Thermo Fisher Scientific Cat#28908) in PBS for overnight at 4°C. After removing the head and tail using micro scissors, larvae were permeabilized by subsequent incubation in acetone and Proteinase K (Invitrogen Cat#25530-049) as described elsewhere [80]. Samples were re-fixed in 4% PFA in PBS and Click-iT TUNEL Alexa Fluor 647 Imaging Assay (Thermo Fisher Cat#C10247) was performed according to the manufacturer's protocol to label apoptotic cells.…”

Section: Methodsmentioning

confidence: 99%

Highly Nonlinear Dynamics of In Vivo Deep-Tissue Interaction with Femtosecond Laser Pulses at 1030 nm

Fattahi,

Jun,

Herbst

et al. 2023

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We report on the highly nonlinear behavior observed in the central nervous system tissue of zebrafish (Danio rerio) when exposed to femtosecond pulses at 1030 nm. At this irradiation wavelength, photo damage becomes detectable only after exceeding a specific peak intensity threshold, which is independent of the photon flux and irradiation time, distinguishing it from irradiation at shorter wavelengths. Furthermore, we investigate and quantify the role of excessive heat in reducing the damage threshold, particularly during high-repetition-rate operations, which are desirable for label-free and multi-dimensional microscopy techniques. To verify our findings, we examined cellular responses to tissue damage, including apoptosis and the recruitment of macrophages and fibroblasts at different time points post-irradiation. These findings substantially contribute to advancing the emerging nonlinear optical microscopy techniques and provide a strategy for inducing deep-tissue, precise and localized injuries using near-infrared femtosecond laser pulses.

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Mechanical spinal cord transection in larval zebrafish and subsequent whole-mount histological processing

Cited by 8 publications

References 18 publications

Small leucine-rich proteoglycans inhibit CNS regeneration by modifying the structural and mechanical properties of the lesion environment

Small leucine-rich proteoglycans inhibit CNS regeneration by modifying the structural and mechanical properties of the lesion environment

Optimizing spinal cord injury in zebrafish larvae: effects of age on the injury response

Highly Nonlinear Dynamics of In Vivo Deep-Tissue Interaction with Femtosecond Laser Pulses at 1030 nm

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