Depletion of Ly6G/Gr-1 leukocytes after spinal cord injury in mice alters wound healing and worsens neurological outcome

Abstract

Spinal cord injury (SCI) induces a robust inflammatory response and the extravasation of leukocytes into the injured tissue. To further knowledge of the functions of neuroinflammation in SCI in mice, we depleted the early arriving neutrophils using an anti-Ly6G/Gr-1 antibody. Complete blood counts revealed that neutrophils increased approximately 3-fold over uninjured controls and peaked at 6-12 h after injury, and that anti-Ly6G/Gr-1 treatment reduced circulating neutrophils by >90% at these time points. Intravital and spinning disk confocal microscopy of the exposed posterior vein and postcapillary venules showed a significant reduction in rolling and adhering neutrophils in vivo after anti-Ly6G/Gr-1 treatment; this was accompanied by a parallel reduction in neutrophil numbers within the injured spinal cord at 24 and 48 h as determined by flow cytometry. The evolution of astrocyte reactivity, a wound healing response, was reduced in anti-Ly6G/Gr-1-treated mice, which also had less spared white matter and axonal preservation compared with isotype controls. These histological outcomes may be caused by alterations of growth factors and chemokines important in promoting wound healing. Importantly, anti-Ly6G/Gr-1 treatment worsened behavioral outcome as determined using the Basso Mouse Scale and subscores. Although the spectrum of cells affected by anti-Ly6G/Gr-1 antibody treatment cannot be fully ascertained at this point, the correspondence of neutrophil depletion and worsened recovery suggests that neutrophils promote recovery after SCI through wound healing and protective events that limit lesion propagation.

Figure 1.

A–D, Anti-Ly6G/Gr-1 treatment reduces circulating neutrophils after SCI. A, Absolute neutrophil counts from whole blood collected from naive (uninjured; gray bar) mice and at 6, 12, and 48 h after SCI reveal a pronounced neutrophilia peaking at 6–12 h after injury (isotype animals; black bars in graphs). Anti-Ly6G/Gr-1 treatment (white bars) markedly (p < 0.001, ANOVA) depleted neutrophils from 6 to 48 h after injury. In contrast, SCI induced a reduction in circulating monocytes (B) and lymphocytes (C), but their numbers were not significantly altered by anti-Ly6G/Gr-1 treatment. WBC counts were reduced in neutrophil-depleted animals (D). Data are represented as mean × 10⁶ cells/ml ± SEM, ANOVA with Holm-Sidak multiple comparisons test. ^#p < 0.05 naive vs all groups except isotype 48 h (A). ^†p < 0.001 naive vs all anti-LyG6/Gr-1 groups and ^‡p < 0.001 naive vs isotype 48 h SCI (D). *p < 0.05, **p < 0.01, ***p < 0.001, n = 3–6 per group, per time point.

Figure 2.

Anti-Ly6G/Gr-1 treatment reduces neutrophil rolling and adhesion on spinal vasculature after SCI. A, Micrographs collected from a time-lapse recording of exposed spinal cord vasculature from an uninjured (sham) mouse revealing a rhodamine G-labeled leukocyte (white arrows) slowing down and rolling along a blood vessel lumen over time (seconds) is shown in the top right corner and velocity (micrometers per second) in the bottom left corner of images. Few leukocytes are seen rolling in uninjured (sham) animals. Scale bar, x, y: 100 μm. B, In contrast, at 6 h after SCI, many leukocytes are present within the posterior vein (shown) and postcapillary venules in isotype-treated animals (top) compared with anti-Ly6G/Gr-1-treated animals (bottom). Scale bar, x, y: 100 μm. C, Quantification of rolling flux (cells/min/100 μm) (top) and firm adherence (cells stationary for 30 s or longer/100 μm²) (bottom) reveals that anti-Ly6G/Gr-1 treatment significantly (p < 0.001, ANOVA) reduces both neutrophil rolling and firm adhesion at both 6 and 12 h after injury. D, A higher magnification representative image of the exposed spinal cord vasculature from video captured at 6 h after SCI using spinning disk confocal microscopy. Note that the majority of rhodamine 6G-positive leukocytes (red channel) used for quantification in C are indeed neutrophils (Gr-1 positive, blue channel), Scale bar: 20 μm. At 48 h after injury, anti-Ly6G/Gr-1 treatment (E, right) markedly reduced the number of neutrophils (Gr-1, blue channel) compared with isotype controls (E, middle) interacting with the endothelium (PECAM-1, green channel). In contrast, few neutrophils were present in uninjured blood vessels (E, left). Data are represented as mean ± SEM per group, ANOVA with Holm-Sidak multiple comparison test. **p < 0.01, *p < 0.05, ^#p < 0.05 sham versus all other groups; ^†p < 0.05, 15 min after SCI versus 6 and 12 h after SCI isotype controls, n = 3–5 per group.

Figure 3.

A–M, Anti-Ly6G/Gr-1 treatment reduces neutrophil accumulation within the injured spinal cord. Representative density plots of injured spinal cord cells at 24 h (A) and 48 h (B) after injury. Microglia (CD45^low:CDllb⁺, boxed region R3), and blood-derived myeloid cells (CD45^high:CDllb⁺, boxed region R2), are differentiated using flow cytometry. Further separation of neutrophils (CD45^high:CDllb^highGr-1^high) is shown in R5. The percentage of each population is indicated. As shown in A and B, neutrophils (R5) are clearly reduced at 24 and 48 h after SCI in anti-Ly6G/Gr-1-treated mice (bottom) versus isotype controls (top). C, Quantification of blood-derived myeloid cells entering the spinal cord reveals that the percentage of neutrophils is significantly (p < 0.001) reduced at both 24 and 48 h after injury in neutrophil-depleted mice (white bars) compared with isotype control animals (black bars). Data are represented as mean percentage ± SEM, n = 4–5 per group, ANOVA. ***p < 0.001. Lysozyme-GFP mice reveal that fewer myeloid cells (green) enter the injured spinal cord in neutrophil-depleted mice (E) compared with isotype controls (D) at 24 h after SCI. Scale bar, 200 μm. Higher magnification images show several myeloid cells (GFP, green) in close contact to Iba-1-positive (red) microglia/macrophages (F) in isotype animals, whereas fewer myeloid cells are present in neutrophil-depleted mice (G). Scale bar, 10 μm. H–M, Greater numbers of neutrophils are evident within the injured spinal cord at 48 h after SCI in isotype control animals using the neutrophil-specific marker 7/4 (red, H) compared with neutrophil-depleted mice (K). Merged images of neutrophils in neutrophil-depleted injured spinal cord lesions (M) and isotype animals (J) are shown. The nuclear stain Hoechst (blue) reveals the polymorphic nucleus characteristic of neutrophils (I, L; J, inset). Scale bar: (J) for H–M: 20 μm; for J, inset: 10 μm.

Figure 4.

Anti-Ly6G/Gr-1 treatment alters cytokine and growth factor levels within the injured spinal cord at 48 h and 5 d after SCI. A, Quantification of inflammatory protein levels isolated from the injured spinal cord at 48 h after injury reveals a significant increase in MIP-1γ/CCL9, KC/CXCL1, G-CSF, and MCP-1/CCL2 protein levels in neutrophil-depleted animals (white bars) versus isotype (black bars) or sham controls (gray bars). Data are represented as mean protein levels [arbitrary units (A.U.)] ± SEM, n = 4 per group. ANOVA, Holm-Sidak method. *p < 0.05, isotype versus α-Ly6G/Gr-1-treated animals. ^#p < 0.05, sham versus isotype. ^†p < 0.05 sham versus α-Ly6G/Gr-1-treated animals. Analysis of growth factor mRNA levels isolated from the injured spinal cord at 48 h (B) and 5 d (C) after SCI using quantitative RT-PCR arrays. Plots of growth factor gene expression with a threefold change or higher are displayed, with α-Ly6G/Gr-1-treated (y-axis) and isotype controls (x-axis) in B and C. Upregulated genes are shown in red, whereas downregulated genes are shown in green. MCP-1/CCL2 monocyte chemoattractant protein-1, KC/CXCL1 keratinocyte derived chemokine, G-CSF granulocyte colony stimulating factor, MIP-1γ/CCL9, macrophage inflammatory protein-1 gamma, Bone morphogenic protein (BMP), fibroblast growth factor (FGF), Interleukin (IL), neutrotrophic factor (NTF), Transforming Growth factor (TGF), vasculature endothelial growth factor (VEGF).

Figure 5.

Anti-Ly6G/Gr-1 treatment dampens the evolution of astrocyte reactivity after SCI. A, In sham-injured controls fibronectin (green) is mainly localized to blood vessels and connective tissue as expected. The medial aspect of the ventral column is shown (arrow). Low levels of GFAP (red) delineate astrocytes. In contrast, at 48 h (B, C) and 5 d (D, E) after injury, the evolution of the glial scar is apparent and dense, as revealed using GFAP immunoreactivity in isotype animals (B, D), but much less reactivity is seen in anti-Ly6G/Gr-1 neutrophil-depleted mice (C, E). Asterisks mark the area of maximum GFAP density. Fibronectin staining is indistinguishable between the two groups. Scale bar, 25 μm. Higher magnification images show Iba-1 (green)-positive microglia/macrophages invading past the less densely formed glial scar (GFAP, red) in neutrophil-depleted mice (G) compared with isotype controls (F). Scale bar, 10 μm. H, Quantification of the glial scar at 5 d after injury reveal a significant increase in GFAP signal (percentage area) in isotype (filled circles)- versus anti-Ly6G/Gr-1 (open circles)-treated animals. There was not a significant difference in fibronectin density between the groups. Data are represented as mean percentage ± SEM, n = 4 four per group, Mann–Whitney rank sum test, *p < 0.05.

Figure 6.

A, B, Anti-Ly6G/Gr-1 treatment worsens neurological outcome after SCI. Neutrophil depletion (open squares) worsened functional outcome as revealed by BMS (A) and BMS subscores (B) compared with isotype controls (filled squares). C, Individual mice BMS subscore values are shown at 28 d after injury (y-axis, left) (filled squares, isotype; open squares, anti-Ly6G/Gr-1). Percentage of animals to reach a BMS subscore of 5 and higher at 28 d after injury is also shown (y-axis, right) (filled bar, isotype; open bar, anti-Ly6G/Gr-1). Data are represented as mean ± SEM, n = 11–12 per group; RM-ANOVA, Holm-Sidak method. *p < 0.05. **p < 0.01, ***p < 0.001. D, Representative eriochrome cyanine- and neutral red-stained serial sections throughout the lesion site from isotype-treated (top panels) and anti-Ly6G/Gr-1-treated (bottom panels) animals. Note that at the lesion epicenter, more spared white matter (SWM, yellow) and smaller lesions are evident in the isotype-treated animal. Quantification of SWM (E) and lesion size (F) reveals significantly less SWM and greater lesion size in neutrophil-depleted (open circles, individual animals; open square, mean) versus isotype control animals (filled circles; filled square, mean). Data are represented as mean percentage ± SEM, n = 11–12 per group, Mann–Whitney Rank Sum test, *p < 0.05. G–I, NF200 staining of the lesion epicenter from an isotype (G), a neutrophil-depleted (H), or a sham-control animal (I) is shown [(scale bar: (G–I), 500 μm. Less NF200 staining is evident in neutrophil-depleted versus isotype controls (arrows in G and H). Quantification of axonal density (NF200 percentage of area occupied) reveals a significant decrease in NF200 density in neutrophil depleted animals (open bars) versus isotype-treated animals (filled bars). Data are represented as mean ± SEM, n = 11–12 per group, Mann–Whitney Rank Sum test, *p < 0.05.