Study On The Role And Mechanism Of Sympathetic Nerve Activation In Diabetic Keratopathy

Purpose:Diabetic keratopathy(DK)exhibits reduced corneal nerve density,sensitivity,and delayed wound healing.Previous studies have shown sympathetic nerve hyperactivation in diabetic patients,but the relationship of sympathetic nerve activation and DK remains unknown.This study was designed to investigate the role and mechanism of sympathetic nerve in DK.Methods:The repair rate of streptozotin-induced type I diabetic mellitus(DM)mice and normal mice of the same age was observed by fluorescin sodium staining at 0,24,36,and 48 h after corneal epithelium debrided.Corneal sensitivity was measured by Cochet Bonnet esthesiometer,and corneal nerve was analyzed by βIII-tubulin immunofluorescence staining.Plasmic and corneal content of norepinephrine(NE)were detected by ELISA at12 h and 24 h after debridment of corneal epithelium in DM and normal mice.Systemic intraperitoneal application of 6-hydroxydopamine(6-OHDA)ablating sympathetic nerve in DM was used to observe the therapeutic effect of sympathetic denervation on corneal epithelium repair and nerve regeneration.In vivo,after corneal epithelium debrided in normal mice,topical application of NE were used to observe the effects of the repair of corneal epithelium,corneal nerve density and the expression of neurotrophic factors.In vitro,human corneal epithelial cells and murine corneal epithelial stem/progenitor cell line(TKE2)were treated with PBS,NE,NE and β1 adrenergic receptor(ADRB1)inhibitor Atenolol or NE and β2 adrenergic receptor(ADRB2)inhibitor ICI 118,551.The effects of NE and adrenergic receptor inhibitors on the expression of neurotrophic factors in corneal epithelial cells were analyzed by qPCR,ELISA and immunofluorescence staining.The primary mouse trigeminal ganglion(TG)neurons were isolated and cultured in vitro.TKE2 conditioned medium supernatant treated with PBS,NE,NE and atenolol or NE and ICI 118,551 were co-cultured with TG.After 24 h,the growth length of trigeminal nerve was analyzed by βIII-tubulin immunofluorescence staining.Key adrenergic receptor inhibitor was used further to evaluate effects after corneal epithelium debrided in DM.The corneal epithelial repair was observed by fluorescein sodium staining,the corneal nerve repair was analyzed by βIII-tubulin immunofluorescence staining,and the expression of neurotrophic factors in corneal epithelial cells was detected by qPCR and immunofluorescence staining.Results:The DM mice exhibited increased plasmic and corneal NE content,slower repair rate of corneal epithelium,and decreased nerve density and corneal sensitivity compared to normal mice,which were rescued by the treatment of sympathetic neurotoxin 6-OHDA.Moreover,exogenous NE treatment recapitulated the characteristics of DK in healthy mice,accompanied by local depletion of multiple neurotrophic factors,such as nerve growth factor and glial cell–derived nerve growth factor.In the co-culture model of corneal epithelial cells and TG,the conditioned medium of NE-treated TKE2 conditioned medium inhibited TG neurite outgrowth,which was reversed by the ADRB2 antagonist ICI 118,551,but not ADRB1 antagonist.In addition,topical application of ICI 118,551 recovered the expression levels of corneal neurotrophic factors,thus promoting corneal wound healing,corneal nerve regeneration,and sensation recovery in DM mice.Conclusions:Sympathetic nerve hyperactivation accompanying with increased NE release was observed in DM after corneal epithelium debrided.NE further binding to ADRB2 receptor in corneal epithelial cells reduces the expression of corneal neurotrophic factors and inhibits corneal nerve regeneration.Therefore,topical application of ADRB2 inhibitors may effectively improve the expression of corneal neurotrophic factors and provide a new therapeutic strategy for the treatment of DK.