Abstract
Injury to the sciatic nerve leads to degeneration and debris clearance in the area distal to the injury site, a process known as Wallerian degeneration. Immune cell infiltration into the distal sciatic nerve plays a major role in the degenerative process and subsequent regeneration of the injured motor and sensory axons. While macrophages have been implicated as the major phagocytic immune cell participating in Wallerian degeneration, recent work has found that neutrophils, a class of short-lived, fast responding white blood cells, also significantly contribute to the clearance of axonal and myelin debris. Detection of specific myeloid subtypes can be difficult as many cell-surface markers are often expressed on both neutrophils and monocytes/macrophages. Here we describe two methods for detecting neutrophils in the axotomized sciatic nerve of mice using immunohistochemistry and flow cytometry. For immunohistochemistry on fixed frozen tissue sections, myeloperoxidase and DAPI are used to specifically label neutrophils while a combination of Ly6G and CD11b are used to assess the neutrophil population of unfixed sciatic nerves using flow cytometry.
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Acknowledgments
The authors thank Mike Sramkoski of the Cytometry and Imaging Microscopy Core at Case Western Reserve University for providing guidance with flow cytometry setup and analysis and Dr. Franklin Echevarria for assistance with the figures. This work was supported by National Institutes of Health grants DK097223 and NS095017 to R.E.Z. J.A.L. was supported by a training grant NS067431 and an NIH NRSA Award F31NS093694. J.P.N. was supported by training grants NS067431 and NS077888.
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Niemi, J.P., Lindborg, J.A., Zigmond, R.E. (2020). Detection of Neutrophils in the Sciatic Nerve Following Peripheral Nerve Injury. In: Babetto, E. (eds) Axon Degeneration. Methods in Molecular Biology, vol 2143. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0585-1_16
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DOI: https://doi.org/10.1007/978-1-0716-0585-1_16
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