An early granulocyte colony-stimulating factor treatment attenuates neuropathic pain through activation of mu opioid receptors on the injured nerve

Abstract

Several studies have shown that the mu opioid receptor (MOR) located in the peripheral nerves can be activated after nerve injury and that it attenuates peripheral nociceptive signals to the spinal dorsal horn. Various cytokines and phosphorylated-p38 (p-p38) activation in the dorsal horn also play an important role in neuropathic pain development. Granulocyte-colony stimulating factor (GCSF) is a growth factor that can stimulate granulocyte formation and has been shown to exert an analgesic effect on neuropathic pain through recruiting opioid-containing leukocytes to the injured nerve. However, the underlying mechanisms are not well understood. Herein, the results of behavior tests in addition to MOR levels in the injured sciatic nerve and the levels of p-p38 and various cytokines in the spinal dorsal horn were studied in vehicle-treated or GCSF-treated chronic constriction injured (CCI) rats at different time points (i.e., 1, 3, and 7 days, respectively) after nerve injury. The results showed that a single early systemic GCSF treatment after nerve injury can up-regulate MORs in the injured nerve, which can decrease peripheral nociceptive signals. Thereafter, those changes suppress the pro-inflammatory cytokine IL-6 but enhance the anti-inflammatory cytokine IL-4, followed by decreases in p-p38 in the dorsal horn, and thus further attenuate neuropathic pain.

Figure 1. GCSF treatment alleviates mechanical allodynia in rats with CCI.

Statistically significant mechanical allodynia developed in the vehicle-treated CCI rats, compared to the sham-operated controls from the 1^st to the 7^th day after nerve injury (P < 0.01). In contrast, early GCSF treatment alleviated mechanical allodynia from the 1^st to the 7^th day after nerve injury compared to the vehicle-treated CCI rats (P < 0.01). The data are shown as the mean ± SEM. Two-way repeated measures ANOVA followed by post hoc LSD test. ^##P < 0.01: vehicle-treated groups compared to sham-operated controls. **P < 0.01: GCSF-treated CCI groups compared to vehicle-treated CCI groups (n = 7, in each group).

Figure 2. GCSF increased mu opioid receptor (MOR) levels in the injured sciatic nerve.

The MOR levels in the injured sciatic nerve, as determined by western blot analysis, were significantly higher in the GCSF-treated CCI rats than in the vehicle-treated CCI rats from the 1^st to the 3^rd day after nerve injury (P < 0.05). The data are shown as the mean ± SEM. ^#P < 0.05, ^##P < 0.01: GCSF-treated groups compared to sham-operated controls. *P < 0.05, **P < 0.01: GCSF-treated CCI groups compared to vehicle-treated CCI groups. One-way ANOVA, post hoc LSD test or Kruskal–Wallis, post hoc Mann–Whitney rank-sum test, if appropriate (n = 5, in each group).

Figure 3. GCSF increased mu opioid receptor (MOR)-positive axons in the injured sciatic nerve.

Representative images of double immunofluorescence for MOR (red) and calcitonin gene related peptide (CGRP), a marker of peptidergic small to medium size neurons (green; (A)); NF200, heavy subunit of neuro-filament (green; (B)); and S100, β-subunit of Schwann cell (green; (C)) in the injured sciatic nerve of the sham controls, vehicle-treated CCI rats, and GCSF-treated CCI rats on the 3rd day after nerve injury. (D) Quantification of MOR-positive axons in the injured sciatic nerve of sham controls, vehicle-treated CCI, and GCSF-treated CCI rats. A significantly higher number of MOR-positive axons were observed in GCSF-treated CCI rats than in vehicle-treated CCI rats (P < 0.05). The MOR-positive axons in the injured sciatic nerve were also slightly higher in vehicle-treated CCI rats than in the sham control rats (P < 0.05). Most MORs were co-stained with CGRP-positive axons and S100-positive Schwann cells. There were only a few MOR-positive stained cells co-localized with N200-positive axons. Scale bars = 20 μm. The data are shown as the mean ± SEM. ^#P < 0.05, ^##P < 0.01: GCSF-treated or vehicle-treated groups compared to sham-operated controls. *P < 0.05, **P < 0.01: GCSF-treated CCI groups compared to vehicle-treated CCI groups. Kruskal–Wallis, post hoc Mann–Whitney rank-sum test (n = 4, in each group). Arrowheads indicate CGRP-positive/MOR-positive axons. Some S100-positive/MOR-positive cells were also observed.

Figure 4. GCSF decreased the pro-inflammatory cytokine IL-6 but increased the anti-inflammatory cytokine IL-4.

(A) ELISA study revealed significantly higher pro-inflammatory cytokine IL-6 levels in the dorsal horn of the vehicle-treated CCI rats than in the sham controls (P < 0.01) from the 1^st to the 7^th day after nerve injury. In contrast, significantly lower IL-6 levels were observed in the dorsal horn of GCSF-treated CCI rats than in the vehicle-treated CCI rats from the 1^st to the 7^th day after nerve injury (P < 0.05). (B) ELISA study revealed significantly lower anti-inflammatory cytokine IL-4 levels in the dorsal horn of the vehicle-treated CCI rats than in the sham controls on the 3^rd day after nerve injury (P < 0.01). In contrast, significantly higher IL-4 levels were observed in the dorsal horn of GCSF-treated CCI rats than in the vehicle-treated CCI rats from the 1^st to the 7^th day after nerve injury (P < 0.05). The data are shown as the mean ± SEM. ^#P < 0.05, ^##P < 0.01: GCSF-treated or vehicle-treated groups compared to sham-operated controls. *P < 0.05, **P < 0.01: GCSF-treated CCI groups compared to vehicle-treated CCI groups. One-way ANOVA, post hoc LSD test (n = 5, in each group).

Figure 5. GCSF decreased the p-p38/total p38 ratio in the spinal dorsal horn.

The phosphorylated-p38/total p38 ratio in the spinal dorsal horn, as determined by western blot analysis, was significantly higher in vehicle-treated CCI rats than in sham controls on the 3^rd day after nerve injury (P < 0.01). In contrast, a significantly lower p-p38/total p38 ratio was observed in the dorsal horn of GCSF-treated CCI rats than in vehicle-treated CCI rats on the 3^rd day after nerve injury (P < 0.01). The data are shown as the mean ± SEM. ^#P < 0.05, ^##P < 0.01: vehicle-treated groups compared to sham-operated controls. *P < 0.05, **P < 0.01: GCSF-treated CCI groups compared to vehicle-treated CCI groups. One-way ANOVA, post hoc LSD test or Kruskal–Wallis, post hoc Mann–Whitney rank-sum test, if appropriate (n = 6, in each group).

Figure 6. GCSF decreased p-p38-positive cells in the dorsal horn.

Representative images of double immunofluorescence in the dorsal horn for p-p38 (red) and OX-42, a microglia marker (green; (A)); GFAP, an astrocyte marker (green; (B)); and NeuN, a neuronal marker (green; (C)) of the sham controls, vehicle-treated CCI rats, and GCSF-treated CCI rats on the 3^rd day after nerve injury. Most p-p38 was co-stained with OX-42-positive cells. (D) Quantification of the OX-42/p-p38-positive cells in the right quarter part of the spinal dorsal horn (lamina I–V) of the sham controls, vehicle-treated CCI, and GCSF-treated CCI rats. The vehicle-treated CCI rats had a significantly higher number of OX-42/p-p38-positive cells than the sham control rats on the 3^rd day after nerve injury (P < 0.01); in contrast, the GCSF-treated CCI rats had a significantly lower number of OX-42/p-p38-positive cells than the vehicle-treated CCI rats on the 3^rd day after nerve injury (P < 0.01). Scale bars = 20 μm. The data are shown as the mean ± SEM. ^#P < 0.05, ^##P < 0.01: GCSF-treated or vehicle-treated groups compared to sham-operated controls. *P < 0.05, **P < 0.01: GCSF-treated CCI groups compared to vehicle-treated CCI groups. Kruskal–Wallis, post hoc Mann–Whitney rank-sum test (n = 5, in each group). Arrowheads indicate OX-42-positive/p-p38-positive cells.

Figure 7. The time sequence of the effects of G-CSF on MOR levels in the injured nerve and of the cytokine levels and microglial/p-p38 activation in the spinal dorsal horn.

Compared to vehicle-treated CCI rats, the sequential effects of GCSF on GCSF-treated CCI rats increased opioid containing polymorphonuclear (PMN) cells from 12 to 48 hours and increased mu opioid receptor (MOR) levels on days 1–3 at the nerve ligature site after nerve injury; decreased IL-6 and TNF-α levels on days 2–6, and days 3–6, respectively, in the dorsal root ganglion (DRG) after nerve injury; decreased IL-6 but increased IL-4 levels from days 1–7 in the spinal dorsal horn (SDH) after nerve injury; and suppressed microglia and p-p38 activation on days 3–6 and on day 3, respectively, in the spinal dorsal horn after nerve injury. (▼: Opioid, : Mu opioid receptor [MOR], : IL-6, : IL-4, ↑: increase, ↓: decrease.)