| Purpose This project aims to establish a form-deprivation myopia model in BN rats and investigate the preventive effects of atropine eye drops at different concentrations on the development and progression of myopia and the possible mechanism.Additionally,LC-MS/MS analysis was used to measure atropine concentrations in plasma and various tissues of rabbit eyes at different time points after eye drop administration to explore the pharmacokinetics of atropine in the eye and whole body,further supporting the discussion on the possible mechanism and potential system toxicity effect.MethodEstablishment of a Form-Deprivation Myopia Model in BN Rats: After anesthesia,disinfection,and care for each animal,an opaque eye occluder was sutured to the skin around the left eye using non-absorbable sutures and completely covered the left eye.A plastic collar was fitted to prevent the rat from removing the eye occluder.Fifty(50)μL of atropine eye drops at the appropriate concentration was drawn and instilled into the left eye using an insulin injection needle.Animals in the blank control group and model control group were given the same volume of polyvinyl alcohol eye drops in the same manner as those in the treatment groups,once a day for seven weeks.After model establishment and drug administration,the axial length,refractive error,ERG,retinal and choroidal blood flow of all animals were detected using A-scan ultrasound,slit-lamp microscopy,Diagnosys Celeris animal electrophysiology instrument,fundus camera,and OCTA.The thickness of the sclera was measured using HE staining.The gene and protein expression levels of HIF-1α,VEGF,α-SMA,m TOR,Epo-1,and TGF-β in the retina and sclera were measured using immunofluorescence staining,RT-q PCR analysis,and Western blot analysis.Following a single administration of 50 μL 0.05% atropine to each eye of the Chinchilla rabbits,plasma,cornea,aqueous humor,iris,lens,vitreous body,retina,choroid,sclera,and conjunctiva were collected at 0.5 h,1 h,2 h,4 h,24 h,and 72 h after administration.The ocular tissues were homogenized with buffer at a ratio of tissue weight to buffer volume 10 mg to 100 μL.The atropine concentrations in plasma and ocular tissues were determined by LC-MS/MS analysis.ResultsAfter topical administration of 0.01% and 0.025% atropine to form-deprivated eyes for 7weeks,axial elongation and refractive error were alleviated.Moreover,these parameters were less pronounced in the 0.025% dose group than in the 0.01% dose group.Compared with the blank control rats,there was no significant change in retinal thickness in BN rats with form-deprivation myopia,but there was an increasing trend in retinal and choroidal blood flow.After administration,the retinal and choroidal blood flow in both treatment groups decreased compared with the model control group.Compared with the blank control rats,the sclera of myopic rats became thinner,and thickened after drug administration.Under 0.01 cd.s/m light stimulation,the fluctuation amplitudes of a-wave and b-wave in the model control group were smaller than those in the blank control group,with significant differences(P<0.01).Compared with the model control group,the decrease in a-wave amplitude of rats in the 0.01% dose group was significant(P <0.05),and the decrease in a-wave amplitude of rats in the 0.025% dose group was extremely significant(P <0.01);while there was not significant difference in b-wave amplitude(P ≥0.05)in both treatment groups.Under 0.1 cd.s/m light stimulation,the a-wave amplitude in the model control group was significantly smaller than in the blank control group(P <0.01),while there was no significant difference in b-wave amplitude(P ≥0.05).Compared with the model control group,there was no significant difference in a-wave or b-wave amplitude in the 0.01% dose group(P ≥0.05);the decrease in a-wave amplitude of rats in the 0.025% dose group was extremely significantly(P <0.01),and there was a 27%increase tendency in b-wave amplitude(P ≥0.05).At 1.0 cd.s/m light stimulation,there was no significant difference in a-wave or b-wave amplitude between different groups(P≥0.05).There was no significant difference in Ops between different groups under the same light stimulation intensity(P ≥0.05).Compared with the blank control group,significantly higher gene expression of HIF-1α,VEGF,and α-SMA in the retina was noted in the model control group(P <0.01).The gene expression levels of TGF-β,m TOR,and EPO showed no significant difference P ≥0.05),but there was an increasing tendency.Compared with the model control group,significant lower HIF-1α expression in the retina was noted in the 0.01% dose group(P <0.05),and a decreasing trend in the expression of VEGF,α-SMA,TGF-β,and m TOR though no significant difference(P ≥0.05).In the 0.025% dose group,significantly lower expression of HIF-1α,VEGF,α-SMA,and EPO in the retina was noted(P <0.05),and a decreasing trend in the expression of TGF-β and m TOR though no significant difference(P ≥0.05).The protein expression of HIF-1α,VEGF,α-SMA,and TGF-β in the retina of each group in the Western blot analysis was consistent with the results using RT-q PCR.Compared with the blank control group,no significant difference in the gene expression levels of HIF-1α,VEGF,α-SMA,and TGF-β in the sclera(P ≥0.05)was noted in the model control group.Besides a decrease in HIF-1α,the expression levels of VEGF,α-SMA,and TGF-β showed an increasing trend.Compared with the model control group,though no significant difference in the gene expression levels of VEGF,α-SMA,and TGF-β in the sclera(P ≥0.05)in both the 0.01% dose group and the 0.025% dose group,a decreasing trend was noted,which became more evident as concentration increased.Compared to the blank control group,significantly higher gene expression of HIF-1α,VEGF,and α-SMA in the sclera was noted in the model control group(P <0.05).Compared with the model control group,a relative decrease in the protein expression ofα-SMA in the sclera was noted in the 0.01% dose group,and a decreasing trend was also noted in the HIF-1α and VEGF levels(P ≥0.05).Compared with the model control group,a significant decrease in the protein expression levels of HIF-1α and α-SMA in the sclera(P <0.05)was noted in the 0.025% dose group,and there was also a decreasing tendency of VEGF(P ≥0.05).In the LC-MS/MS assay for the determination of atropine in cyanotic blue rabbits,the lower limit of quantification was 0.5 ng/m L using liquid phase gradient elution and toluenesulfonylurea as the internal standard.After administering 0.05% atropine eye drops to the Chinchilla rabbits,in half an hour to one hour,the distribution of atropine was the highest on cornea,and the rest were iris,aqueous humor,choroid,conjunctiva,sclera and lens,while atropine was not detected in the vitreous and retina.At 2-4 h,the relationship between atropine content in each tissue was: iris> cornea>choroid> aqueous humor> conjunctiva> lens> sclera> retina,vitreous(not detected);After 4 h,the relationship between atropine content in each tissue was as follows: iris>choroid> cornea>lens> sclera,conjunctiva> retina,vitreous(not detected).the peak concentration of atropine in the plasma occurred before 1 hour after administration and then decreased with time until it was below the detection limit,while its maximum content accounted for about 1/25 of the highest content on the cornea.The peak concentrations of atropine in the cornea,aqueous humor,conjunctiva,and sclera of the rabbits occurred before 0.5 hours after administration.The peak concentrations of atropine in the vitreous body and retina occurred between 0.5-2 hours after administration,while the peak concentrations in the iris and choroid occurred between 2-4 hours after administration.The peak concentration of atropine in the lens occurred between 1-4 hours after administration.The longest retention time of Atropine was noted on the iris and choroid,up to 72 hours,suggesting that atropine may act not only on the ciliary muscle in the anterior segment of the eye,but also on the choroid,thereby delaying myopia progression.Conclusion1.Myopia can be induced in BN rats by wearing an opaque eye occluder,which is manifested as axial elongation,refractive error,and reduced amplitudes of the a-wave and b-wave in electroretinogram.2.Administering atropine eye drops during the induction of myopia can prevent further progression,and the effect of 0.025% atropine eye drops is better than that of 0.01%.Atropine eye drops can slow down the abnormal increase in retinal and choroidal blood flow during myopia development.Moreover,it can reduce scleral extensibility and maintain scleral thickness within the normal range possibly by reducing the gene and protein expression of HIF-1α,VEGF,α-SMA,and TGF-β in the retina and sclera,further preventing the progression of myopia.3.Following topical administration of 0.05% atropine as eye drops,it can stay in the iris and choroid for at least 72 hours.Based on the OCTA results of the choroidal blood flow in each group,we believe that atropine may also act on the choroid,but the underlying mechanism is still unclear and requires further research. |
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