| Purpose:Keratoconus(KC)is a blinding eye disease with a central cornea thinning and cone-like protrusion occurring during puberty.Corneal cross-linking(CXL)is a treatment that prevents the progression of the keratoconus by increasing corneal biomechanical stiffness.CXL uses photosensitizers riboflavin to produce a large number of reactive oxygen species under the irradiation of ultraviolet rays A(UVA),which can cause type II photochemical reactions between the amino groups(groups)of collagen fibers,thus increasing the corneal stiffness.Currently,the CXL technique is classified as Standard(epi-off)CXL and transepithelial(epi-on)CXL according to whether the corneal epithelium is removed or not.Standard(epi-off)CXL is effective but easy to cause multiple complications such as infection.Transepithelial(epi-on)CXL preserves the corneal epithelium,thus is safer.However,due to the corneal epithelial barrier,riboflavin penetration into the corneal stroma is very limited,and the cross-linking effect is inferior to that of Standard(epi-off)CXL.The effective opening of the corneal epithelial barrier to allow riboflavin to penetrate into the corneal stroma is one of the key techniques for transepithelial CXL.At present,benzalammonium chloride(BKC)and ethylenediamine tetraacetic acid(EDTA)are often used in clinical work to open the corneal epithelial barrier.If the concentration is too low,the barrier opening is very limited,and if the concentration is high,the side effects are greater.Therefore,it is necessary to explore safer and more effective riboflavin penetration enhancers.Norepinephrine(NE)has been reported to affect the integrity of corneal epithelial barrier function.The purpose of this study was to investigate the safety and feasibility of NE as an enhancer promoting corneal penetration of riboflavin in transepithelial CXL applications.Methods:1 Safety study of low dose norepinephrine(2μL)on ocular surfaceC57BL/6 mice were subconjunctivally injected with 2μL,2.5μL,and 3μL of norepinephrine to observe the changes of the ocular surface by slit-lamp.2μL NE was determined and injected into the right eye of mice by subconjunctival injection as the experimental group,and the other left eye was injected with equal dose of Na Cl as the control group.The changes of ocular surface at 3,8,15 and 24 h were observed by slit-lamp.In addition,HE staining and OCT detection were used to observe the changes of corneal histology and corneal thickness respectively.2 Study on the effectiveness of norepinephrine in opening tight junctions between corneal epithelial cells(1)After subconjunctival injection of 2μL NE,the tight junctions of corneal epithelial cells were observed by scanning electron microscopy(SEM)at the 15 h.The recovery state after a long time(5-7 days)was also observed.(2)In the in vitro cell culture experiment,human corneal epithelial cells(HCEC)were treated with NE(10μM)for 1 h,followed by immune response,and finally the changes in ZO-1 protein,a marker of tight junctions between cells,were observed.Na Cl-treated HCEC were used as controls for comparison.3 Study on norepinephrine promoting riboflavin infiltration into corneal stromaTo begin with,positive controls were established by directly injecting riboflavin into the stroma and anterior chamber.Subsequently,2μL NE was subconjunctivally injected into eyes,and riboflavin penetration was performed 15 h later.Riboflavin penetration into the corneal stroma was observed 2-3 h later by slit lamp.In addition,the self-made riboflavin storage tank(Simulate the riboflavin storage tank used in the clinical procedure of riboflavin infiltration)assists the temporary storage and uniform penetration of riboflavin on the surface of the cornea.A certain liquid level height makes it have a certain hydraulic effect,also known as hydraulic column.Then the permeability uniformity of riboflavin was observed by confocal microscope 3D scanning.4 Efficacy of low dose(2μL)norepinephrine versus clinical transepithelial drug(1)2μL NE was injected subconjunctival into the ocular surface,which regards as the experimental group.Riboflavin infiltration was performed for 2-3 hrs after 15 h.(2)The clinical transepithelial drug Peschke TE was applied to the ocular surface of mice directly or with the aid of hydraulic column,which regards as control group,and riboflavin penetration was performed for 30 min.(3)After riboflavin penetration,collagen cross-linking reaction was performed in both groups under UVA irradiation with an irradiance of 3 m W/cm~2for 30 minutes.(4)The indentation test was used to detect the biomechanical properties of the two groups of corneas after crosslinking.5 Feasibility study of norepinephrine application in clinical scenarios(1)Norepinephrine was increased to 5.5μL and injected subconjunctivally into the ocular surface as simulate the clinical rapid CXL regimen group(S-CXL group).(2)The connections between epithelial cells were observed by scanning electron microscopy after 8 h.The recovery state after a long time(5-7 days)was observed.(3)8 h later,riboflavin was infiltrated for 30 min,and CXL with an irradiance of 9m W/cm~2for 2.5 min.(4)Corneal biomechanical properties after CXL was detected by indentation instrument,and normal corneas without any treatment were compared as controls.(5)Corneas of the experimental group were collected after CXL,and the corneal endothelium was stained with alizarin red and its morphology and density were analyzed.Normal corneas without any treatment were compared as controls.Results:1 A low dose(2μL)of norepinephrine can be safely used on the ocular surface.(1)The cornea showed slight edema at the 3 h,8 h,and returned to normal at the 15 h.(2)Corneal HE staining showed changes in corneal thickness at different time points.(3)OCT showed cornea thickened at 3 and 8 h,and recovered to the same thickness as Na Cl control group at 15 h.2 Dissociation of corneal epithelial junctions after NE treatment(1)After treated with 2μL NE for 15 h,Corneal epithelial cells dissociated slightly observed by scanning electron microscopy.After a period of observation(5-7 days),it was found that the corneal epithelium recovered to be equivalent to normal(2)In the in vitro cell culture experiment,the protein of ZO-1,a marker of tight junction between human corneal epithelial cells,was reduced and the cell tight junction was dissociated after NE(10μM)treatment.3 Norepinephrine can promote riboflavin penetration into the corneal stroma(1)Under cobalt blue light illumination,if riboflavin was directly injected into the corneal stroma,the anterior chamber of the weak green stripe(riboflavin reflection beam)and iris blue line(reflected light strips on the iris surface under cobalt-blue light)could be seen.If riboflavin was injected directly into the anterior chamber,strong and obvious green stripe was observed in the anterior chamber,but no iris blue line was seen.These status above can be used as positive controls to identify the penetration location of riboflavin.(2)15 h after 2μL NE treatment,riboflavin infiltrating for 2-3 h,the cornea can be seen a green stripe and a iris blue line under cobalt blue light illumination,indicating that the riboflavin has penetrated into the corneal stroma.(3)After confocal microscope observation,it was found that the self-made storage tank could make riboflavin penetration more uniform.4 Low dose(2μL)of norepinephrine could have comparable effects with clinical transepithelial drugs(1)Under cobalt blue light illumination,riboflavin could penetrate into the corneal stroma for 2-3 h after 2μL NE treatment,and riboflavin also reached the corneal stroma after 30 min treatment with the transepithelial drug Peschke TE.(2)After CXL was performed on both groups,the NE treatment group exhibited a lower velocity of stress relaxation and a higher G(∞)compared to the Peschke TE treatment group.However,there was no significant difference observed in the corneal elastic modulus between the two groups.5 High dose(5.5μL)norepinephrine is feasible to be used in clinical settings as an enhancer of riboflavin penetration(1)NE was increased to 5.5μL.After 8 h of treatment,the corneal epithelial cells were separated significantly according scanning electron microscopy examination results.In addation,it can recover to normal after a period of time(5-7 days).(2)The modified NE dose was 5.5μL.After 8 h of treatment,the obvious separation of intercellular connections suggested riboflavin application.After the penetration time of riboflavin was shortened to 30 min,riboflavin could still penetrate into the corneal stroma.(3)Compared with normal cornea group,the stress relaxation rate of S-CXL group was significantly slower,the G(∞)and the elastic modulus of S-CXL group was increased significantly after crosslinking.(4)There was no statistical difference between S-CXL group and normal group in the density of corneal endothelial cells after crosslinking.Conclusions:Based on the results,it can be concluded that the application of low dose norepinephrine on the ocular surface is safe and effective in promoting riboflavin penetration into the corneal stroma.After the modification,high dose of norepinephrine shortened the penetration time of riboflavin significantly,and had no obvious damage to the corneal endothelium after crosslinking,and significantly enhanced the biomechanical properties.Therefore,norepinephrine as an enhancer of riboflavin penetration probably has great potential for clinical application in transepithelial corneal collagen crosslinking. |
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