Detection of Neutrophils in the Sciatic Nerve Following Peripheral Nerve Injury

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.

Keywords: Flow cytometry; Immunohistochemistry; Neutrophil; Peripheral nerve injury; Sciatic nerve; Wallerian degeneration.

Figure 1.. Diagram of the sciatic nerve in the mouse hind limb.

Graphical illustration of the location of the sciatic nerve in the mouse hind limb. Displayed in this figure are the relative site of injury (denoted by a red dashed line), the cut site for dissection of the nerve (denoted by the dashed green line), the major muscles that are located above the sciatic nerve, and the lumbar DRG contribution of sensory axons to the sciatic.

Figure 2.. Representative Images of myeloperoxidase and von Willebrand Factor IHC in a wild-type sciatic nerve 3 days post-injury.

This image, taken at 20x on a Leica SP8 Confocal microscope, displays MPO+ neutrophils (magenta) and VWF+ blood vessels (green) in an injured distal sciatic nerve (A). The staining shows very few neutrophils associated with blood vessels (B; arrowheads). The use of DAPI allows for visualization of the characteristic poly lobed nuclei of neutrophils (C).

Figure 3.. Accumulation of CD11b+Ly6G+ neutrophils in the distal sciatic nerve at 3 and 7 d post-injury measured using flow cytometry.

CD11b+Ly6G- myeloid cells can be distinguished from CD11b+Ly6G+ neutrophils in the sham and injured sciatic nerve (A). Quantification of CD11b+Ly6G- myeloid cells at 3 and 7 d post injury are expressed as a percentage of the total cells detected in the sciatic nerve (B). Quantification of CD11b+Ly6G+ neutrophils at 3 and 7 d post injury are expressed as a percentage of the total cells detected in the sciatic nerve (C). n = 3 samples per group. *p < 0.05. **p < 0.001.