Zinc Improves Functional Recovery by Regulating the Secretion of Granulocyte Colony Stimulating Factor From Microglia/Macrophages After Spinal Cord Injury

Abstract

While zinc promotes motor function recovery after spinal cord injury (SCI), the precise mechanisms involved are not fully understood. The present study aimed to elucidate the effects of zinc and granulocyte colony stimulating factor (G-CSF) on neuronal recovery after SCI. The SCI model was established by Allen's method. Injured animals were given glucose and zinc gluconate (ZnG; 30 mg/kg) for the first time at 2 h after injury, the same dose was given for 3 days. A cytokine antibody array was used to screen changes in inflammation at the site of SCI lesion. Immunofluorescence was used to detect the distribution of cytokines. Magnetic beads were also used to isolate cells from the site of SCI lesion. We then investigated the effect of Zinc on apoptosis after SCI by Transferase UTP Nick End Labeling (TUNEL) staining and Western Blotting. Basso Mouse Scale (BMS) scores and immunofluorescence were employed to investigate neuronal apoptosis and functional recovery. We found that the administration of zinc significantly increased the expression of 19 cytokines in the SCI lesion. Of these, G-CSF was shown to be the most elevated cytokine and was secreted by microglia/macrophages (M/Ms) via the nuclear factor-kappa B (NF-κB) signaling pathway after SCI. Increased levels of G-CSF at the SCI lesion reduced the level of neuronal apoptosis after SCI, thus promoting functional recovery. Collectively, our results indicate that the administration of zinc increases the expression of G-CSF secreted by M/Ms, which then leads to reduced levels of neuronal apoptosis after SCI.

Keywords: G-CSF; NF-kappa B; microglia/macrophages; spinal cord injury; zinc.

Figure 1

Granulocyte colony stimulating factor (G-CSF) was the highest elevated factor among the increasing cytokine profile in spinal cord tissue of acute spinal cord injury (SCI) mice after zinc treatment. The doublet spots in the membranes indicate each cytokine. (A) Intracellular proteins from spinal cord tissue were detected by a cytokine antibody array 1 day after glucose and ZnG treatment. (B) Densitometric analysis of (A; n = 3 per group). (C) The cytokine antibody array was repeated 3 days after glucose and ZnG treatment. (D) Densitometric analysis of (C; n = 3 per group). The expression of G-CSF in the spinal cord was verified by Western blotting (n = 6 per group, Mann-Whitney U test; E,F) and Quantitative real-time PCR (qRT-PCR; n = 6 per group, one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test; G). Data are expressed as means ± standard deviation (SD). **p < 0.01, ***p < 0.001.

Figure 2

Elevated G-CSF was predominantly secreted by microglia/macrophages (M/Ms) after Zinc treatment during the acute stages of SCI. (A) The cellular localization of G-CSF was assessed by double immunofluorescent staining of G-CSF with NeuN, GFAP, Iba-1 and O4, respectively. Two transverse sections were taken from each animal at two points: +4 mm and −4 mm (n = 3 per group, white arrows: G-CSF-positive M/Ms). (B–E) The purity of M/Ms in each SCI lesion was determined by flow cytometry before and after isolation using the magnetic beads sorting method (n = 3 per group, Mann-Whitney U test). The purity of M/Ms was as high as 96% by sorting, meaning that such samples could serve in subsequent experiments. (F) The expression of G-CSF in M/Ms from each SCI lesion site was evaluated by qRT-PCR at the transcriptional level (n = 7 per group, Mann-Whitney U test). Data are expressed as means ± SD, **p < 0.01, ***p < 0.001.

Figure 3

Zinc promoted M/M’s to directly express G-CSF. (A) The cellular viability of Raw264.7 cell was measured by MTT after exposure to different concentrations of ZnG (n = 4). (B) The expression of G-CSF by Western blotting. (C) Quantitative analysis of G-CSF expression. Data are expressed as means ± SD (n = 6 per group, Mann-Whitney U test). **p < 0.01.

Figure 4

Zinc promoted the activation of nuclear factor-kappa B (NF-κB) and its entry into the nucleus. (A) Immunofluorescence staining demonstrated that NF-κB p65 underwent nuclear translocation upon ZnG stimulation in RAW264.7 cells. (B) The expression of G-CSF in the nucleus, as demonstrated by Western blotting. (C) Quantitative analysis of NF-κB expression in the nucleus of cells from the Vehicle and ZnG groups (n = 7 per group, Mann-Whitney U test). Data are expressed as means ± SD. **p < 0.01.

Figure 5

Zinc increased the expression of G-CSF via the NF-κB signaling pathway. Western blotting of indicated proteins at the SCI lesion site (A,C) or in Raw264.7 cells (B,D; n = 6 per group, one-way ANOVA followed by Bonferroni’s post hoc test). (E) Detection of the effect of NF-κB on G-CSF at the mRNA level in M/Ms after SCI, as determined by qRT-PCR. Data are expressed as means ± SD (n = 7 per group, Kruskal-Wallis test). *p < 0.05, ***p < 0.001.

Figure 6

Zinc treatment promoted motor functional recovery by G-CSF following SCI. (A) The neutralizing effect of neutralizing antibody against G-CSF in SCI by Western blotting. (B) Quantitative analysis of G-CSF expression in different groups (n = 6 per group, one-way ANOVA followed by Bonferroni’s post hoc test). (C) Statistical analysis of motor functional recovery of Sham, SCI-Vehicle, SCI-ZnG and SCI-ZnG+GCSF Ab mice before and after SCI using Basso Mouse Scale (BMS) scores (n = 4–5 per group, two-way ANOVA followed by Tukey’s post hoc test). (D) Representative sections showing normal neurons that contained prominent nucleoli, loose chromatin, and Nissl bodies. (E) Quantitative analysis of motor neurons in the ventral horn at 21 days post-injury (n = 6 per group, one-way ANOVA followed by Bonferroni’s post hoc test). Data are expressed as means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

Zinc treatment reduced apoptosis in spinal cord tissue by G-CSF after SCI. (A) Transferase UTP Nick End Labeling (TUNEL)/4′,6-diamidino-2-phenylindole (DAPI) labeling was used to count the number of TUNEL-positive cell in the SCI-Vehicle, SCI-ZnG and SCI-ZnG+GCSF Ab groups. (B) Quantification analysis of the proportion of TUNEL-positive cells in each group (n = 6 per group, one-way ANOVA followed by Bonferroni’s post hoc test). (C) Representative western blots for Bax and Bcl-2 expression, and the loading control (β-Tublin), in SCI-Vehicle, SCI-ZnG and SCI-ZnG+GCSF Ab groups at 3 days post-injury. (D,E) Quantification analysis of the expression levels of Bax and Bcl-2 (n = 6 per group, one-way ANOVA followed by Bonferroni’s post hoc test). Data are expressed as means ± SD, **p < 0.01, ***p < 0.001.

Figure 8

Zinc treatment reduced neuronal apoptosis by G-CSF after SCI. (A) Representative double immunofluorescent staining for Cleaved-caspase-3-positive cells (red), NeuN (green), and DAPI (blue) in SCI of SCI-Vehicle, SCI-ZnG and SCI-ZnG+GCSF Ab mice at 21 days post-injury. (B) Quantification of Cleaved-caspase-3-positive cells co-labeled with NeuN to reveal apoptotic neurons (n = 6–7 per group, one-way ANOVA followed by Bonferroni’s post hoc test). Data are expressed as means ± SD, **p < 0.01, ***p < 0.001.