Roles And Mechanisms Of Neuropeptide Substance P In Mediating Corneal Nerve Regeneration Through Modulating Neutrophils

PurposeTo explore the role and molecular mechanism of SP-NK1 R signaling pathway in corneal nerve regeneration through C57 and B6.Cg-Tac1tm1Bbm/ J(SP KO)mouse corneal nerve injury model.MethodsSP knockout mice were identified by mouse tail gene identification.Slit lamp microscope was used to observe SP knockout mouse corneal phenotype.Corneal nerve distribution was observed by corneal nerve staining.The corneal epithelial injury model was established,the repair rate of corneal epithelial injury was observed by using corneal fluorescein sodium staining;the expression of neurotrophic factor in corneal epithelium was by real-time PCR(RT-PCR).The corneal epithelial injury model was established,the mice were randomly divided into normal control group,normal +L733060 group(subconjunctival injection),SP knockout mice group,SP knockout mice + Sar-SP group(local application).The density of regenerated nerve fibers of mice was observed by corneal nerve staining.The number of chemotaxis of neutrophils and macrophages was analyzed by flow cytometry.The expression of genes related to nerve regeneration after corneal injury in mice was detected by RT-PCR.The changes of corneal neutrophil chemotaxis in mice cornea were verified by immunofluorescence staining.The expression level of NGF in the cornea was detected by enzyme-linked immunosorbent assay(ElISA).The regenerated nerve fiber density the repair rate of corneal epithelial were detected after the neutrophil clearance and macrophage clearance.ResultsMouse tail gene identification showed that Tac-1(SP gene)was completely knocked out.Compared with the control group,the cornea of SP knockout mice was normal,with no spontaneous pathological features in the corneal or corneal innervation(P>0.05).Compared with normal C57 mice,the corneal epithelial repair rate of SP knockout mice was no significant change(P>0.05),and the expression of neurotrophic factors in corneal epithelium of SP knockout mice were no significant change(P> 0.05).Compared with normal mice,the corneal nerve regeneration of SP knockout mouse was significantly reduced(P<0.05),while exogenous Sar-SP can significantly restore the regeneration of the corneal nerve in SP knockout mouse(P<0.05);Subcutaneous injection of L733060 to normal mice could significantly inhibit corneal nerve regeneration(P <0.05).Compared with the control group,the number of chemotaxis of neutrophils(CD45+/CD11b+ / Ly6G+)in SP knockout mouse and L733060 intervention group was significantly reduced(P<0.05),while Sar-SP can significantly restore neutrophil migration,yet the number of macrophages(CD45+/CD11b+/F4/80+)chemotaxis was not affected(P>0.05).Compared with the control group,neutrophils clearence can significantly inhibit corneal nerve regeneration and corneal epithelial healing(P<0.05),yet macrophages depletion has no significant effect on them(P>0.05).Compared with normal mice,the expression of NGF in the cornea of SP knockout mouse was significantly decreased(P<0.05),while exogenous administration of Sar-SP can restore NGF expression(P<0.05).Normal mouse treated with L733060 or removal of neutrophils could significantly reduce NGF expression(P<0.05).The macrophage clearance had no significant effect on the NGF expression(P>0.05).ConclusionSP-NK1 R signaling pathway regulates the regeneration of corneal nerves by affecting the migration of neutrophils and the NGF expression.