Inhibiting EGFR Activation Suppresses Microglial Inflammation And Secondary Damages After Spinal Cord Injury

Objective:Previous reports showed that activated microglia plays a crucial role in the progression of secondary damage after SCI; EGFR phosphorylation is associated with the microglia activation. Thus we aimed to discover the relationship between them, meanwhile, clarify the influence of C225 and AG1478, two EGFR inhibitors, on microglia activation and associated inflammation, furthermore, on the secondary damages and recovery after SCI in rats.Methods:LPS was used to induce the activation of microglias, while weight drop technique was done to induce rat spinal cord trauma. The activated cells and injured rats were treated by either C225 or AG1478. Reverse transcription PCR and ELISA was used to test the mRNA expression and secretion of IL-1βand TNFα, irrespectively. Immunofluorescent staining was utilized to observe the cell morphological change, microglial pEGFR expression, activation of microglias and astrocytes, and blood vessel damages. Western blot was performed to test the expression of EGFR, pEGFR, phosphorylation of Erkl/2, JNK and p38 MAPKs. Calcium image and inhibiting test were performed to show the mechanism of LPS induced EGFR phosphorylation. Wet-dry weight was comparied to show the tissue edema. Axonal tracing and functional scoring were performed to show the recovery of damaged rats.Results:1) C225 and AG1478 attenuated the morphological changes, pEGFR expression in activated microglias and associated expression/secretion of IL-1βand TNFαeffectively.2) Calcium activity in microglias was immediately induced by LPS treatment. EGFR phosphorylation was inhibited by BAPTA/AM and KN62, not EGTA.3) Phosphorylation of Erkl/2, JNK and p38, and expression of IL-1βand TNFa were persistently induced by LPS treatment, which can be attenuated by C225 and AG1478 pre-treatment. Otherwise, specific inhibitors for MAPKs depressed the synthesis/secretion of IL-1βand TNFa in microglias at different degrees.4) Fiercely elevation of pEGFR was shown after-SCI, which can be located in activated microglias.5) Treatment with C225 and AG1478 reduced the expression of pEGFR, phosphorylation of Erkl/2 and p38MAPK, production of IL-1βand TNFa, tissue edema, activation of microglias and astrocytes, morphological changes of blood vessel and formation of glial scar, finally improved the axonal and functional recovery in rats after SCI.Conclusion:Inhibiting EGFR pathway can attenuate the LPS induced microglia activation and associated cytokine production, by depressing the activation of MAPKs. What's more, inhibiting EGFR contributes to depressed inflammation and reduced secondary damages, finally improves the morphological and functional recovery of rats after SCI. EGFR may be a therapeutic target for SCI treatment, and C225/AG1478 is potential to be used to improve the recovery after SCI injury.