Inhibition of EGFR/MAPK signaling reduces microglial inflammatory response and the associated secondary damage in rats after spinal cord injury

Abstract

Background: Emerging evidence indicates that reactive microglia-initiated inflammatory responses are responsible for secondary damage after primary traumatic spinal cord injury (SCI); epidermal growth factor receptor (EGFR) signaling may be involved in cell activation. In this report, we investigate the influence of EGFR signaling inhibition on microglia activation, proinflammatory cytokine production, and the neuronal microenvironment after SCI.

Methods: Lipopolysaccharide-treated primary microglia/BV2 line cells and SCI rats were used as model systems. Both C225 and AG1478 were used to inhibit EGFR signaling activation. Cell activation and EGFR phosphorylation were observed after fluorescent staining and western blot. Production of interleukin-1 beta (IL-1 β) and tumor necrosis factor alpha (TNF α) was tested by reverse transcription PCR and ELISA. Western blot was performed to semi-quantify the expression of EGFR/phospho-EGFR, and phosphorylation of Erk, JNK and p38 mitogen-activated protein kinases (MAPK). Wet-dry weight was compared to show tissue edema. Finally, axonal tracing and functional scoring were performed to show recovery of rats.

Results: EGFR phosphorylation was found to parallel microglia activation, while EGFR blockade inhibited activation-associated cell morphological changes and production of IL-1 β and TNF α. EGFR blockade significantly downregulated the elevated MAPK activation after cell activation; selective MAPK inhibitors depressed production of cytokines to a certain degree, suggesting that MAPK mediates the depression of microglia activation brought about by EGFR inhibitors. Subsequently, seven-day continual infusion of C225 or AG1478 in rats: reduced the expression of phospho-EGFR, phosphorylation of Erk and p38 MAPK, and production of IL-1 β and TNF α; lessened neuroinflammation-associated secondary damage, like microglia/astrocyte activation, tissue edema and glial scar/cavity formation; and enhanced axonal outgrowth and functional recovery.

Conclusions: These findings indicate that inhibition of EGFR/MAPK suppresses microglia activation and associated cytokine production; reduces neuroinflammation-associated secondary damage, thus provides neuroprotection to SCI rats, suggesting that EGFR may be a therapeutic target, and C225 and AG1478 have potential for use in SCI treatment.

Figure 1

EGFR blockade inhibits LPS-induced microglia activation and EGFR phosphorylation. Plated cells were treated with 20nM C225 or 10 μM AG1478 30 min before 1 μg/ml LPS stimulation. After 3 h LPS stimulation, co-staining of CD11b (Green), pEGFR (Red) and nucleus (Blue) was performed. pEGFR expressed in microglias (A1) and BV2 cells (B1) is elevated after LPS stimulation (A2/B2); C225 (A3/B3) or AG1478 (A4/B4) inhibits LPS-induced overexpression of pEGFR. Scale bar = 50 μm. (C) Comparison of cell size suggests that the microglial hypertrophy led by LPS is reduced by C225 or AG1478. *P < 0.01 versus sham; ^#P < 0.01 versus LPS-treated. (D) Western blot analysis of BV2 cells reveals that the LPS-induced upregulation of CD11b and pEGFR is effectively reduced by C225 or AG1478. n = 5. LPS, lipopolysaccharide; pEGFR, phosphorylated epidermal growth factor receptor.

Figure 2

EGFR blockade depresses LPS-induced cytokine production in microglia. Purified microglia was treated with 20nM C225 or 10 μM AG1478 30 min before 1 μg/ml LPS stimulation. (A) Reverse transcriptase-PCR (cells) and (B) ELISA (supernatants) at various time-points. It demonstrates that C225 and AG1478 depress LPS-induced synthesis/secretion of IL-1β and TNFα. n = 5. *P < 0.01 versus LPS-treated. EGFR, epidermal growth factor receptor; LPS, lipopolysaccharide.

Figure 3

MAPK signaling mediates the depression of cytokine production by EGFR blockade. After treated with 20nM C225 or 10 μM AG1478 30 min before 1 μg/ml LPS stimulation, western blot analysis of BV2 cells was performed to show phosphorylation of the MAPKs (Erk, JNK and p38) and cytokine production (IL-1β and TNFα). (A) representative photos. (B) Statistical comparison after normalization to β-actin and its corresponding control. Primary microglia was pretreated with 10 μM selective MAPK inhibitors (SB203580 for p38, U0126 for Erk and SP600125 for JNK) 30 min before LPS stimulation. Synthesis and secretion of cytokines were tested at 3 h and 6 h after LPS stimulation, respectively. (C) and (D) show the representative mRNA expression and analyzed supernatant protein concentrations of IL-1β and TNFα, respectively. n = 5. *P < 0.01 versus LPS-treated. EGFR, epidermal growth factor receptor; Erk, extracellular signal-regulated kinases; JNK, c-jun terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinases.

Figure 4

EGFR phosphorylation is elevated in parallel with microglia activation in early-phase SCI. (A) Representative western blots reveal the elevated expression of pEGFR on days 1 to 14, peaking on day 1. (B) Fluorescent staining of CD11b (Green) demonstrates the gradient activation of microglias on day 3 after SCI. Bar = 500 μm. Double staining of pEGFR (Red) and CD11b reveals that pEGFR⁺ reactive microglias surround the cavity (C) and appear in the boundary zone (D), but not in other areas (E). Bar = 100 μm. Arrow, representative pEGFR⁺ reactive cells. pEGFR, phosphorylated epidermal growth factor receptor; SCI, spinal cord injury.

Figure 5

EGFR blockade reduces EGFR/MAPK activation and cytokine productionin vivo. Continual infusion of C225 or AG1478 was performed immediately after SCI (n = 5). Representative photos of western blots reveal (A) reduced phosphorylated forms of EGFR, Erk, and p38 on day 1, as well as (B) subsequent downregulated expression of IL-1β and TNFα on day 3, after C225 or AG1478 treatment. EGFR, epidermal growth factor receptor; Erk, extracellular signal-regulated kinases; MAPK, mitogen-activated protein kinases.

Figure 6

EGFR blockade attenuates cell activation and tissue edema after SCI. Fluorescent staining reveals (A2 and B2) the activation of microglia and astrocytes on day 7 after SCI, which was attenuated by (A3 and B3) C225 and (A4 and B4) AG1478 treatments. Bar = 200 μM. (C) Similar findings by western blot analysis of CD11b and GFAP 7 day after SCI. (D) Water content of spinal cord reveals that SCI-induced edema was reduced by three days C225/AG1478 treatment. n = 5. ^#P < 0.01 versus sham; *P < 0.01 versus SCI. EGFR, epidermal growth factor receptor; GFAP, glial fibrillary acidic protein; SCI, spinal cord injury.

Figure 7

EGFR blockade promotes morphological and functional recovery. (A2) Combining GFAP staining (red) with BDA tracing (green), fluorescent staining of spinal cord reveals glial scar and cavity formation, as well as limited axonal regeneration after SCI; (A3 and A4) amelioration observed after C225 and AG1478 treatment. Bar = 1 mm. (B) Temporal changes in behavioral outcomes, as evaluated by BBB (B1) and CBS (B2) scoring, indicate that C225 and AG1478 improved functional recovery beginning on day 7 after SCI. n = 7. *P < 0.05 versus SCI. BBB, Basso Beattie and Bresnahan; BDA, biotinylated dextran amine; CBS, combined behavior score; EGFR, epidermal growth factor receptor; GFAP, glial fibrillary acidic protein; SCI, spinal cord injury.