| Objective: Glucocorticoid-induced glaucoma(GIG)is a disease with intraocular pressure(IOP)may be associated with visual field damage after long-term administration of glucocorticoid drugs.Glucocorticoids are widely used in the clinical treatment of various diseases due to their strong anti-inflammatory,anti-allergic and immunosuppressive effects,leading to an increasing incidence of GIG.The mechanism of GIG is until not entirely clear,the current accepted argument is that glucocorticoids could affect the normal growth,metabolism,structure and function of trabecular meshwork(TM),lead to obstruction of aqueous humor discharge caused by increased IOP.11beta-hydroxysteroid dehydrogenase type 1(11β-HSD1)which is known as the " the amplifier of glucocorticoid effect ",is the prereceptor regulator of glucocorticoids at the tissue level.It can activate the inactive cortisone in local tissue into active cortisols,so that increasing the local concentration of activity cortisols.The study was to investigate the relationship between the expression of 11β-HSD1 in ciliary body and TM in the model which was established by use the method of administrtion of subconjunctival injection of0.5% dexamethasone and 0.1% dexamethasone eye drops in New Zealand white rabbits eyes and steroid-induced ocular hypertention.Methods: Fifteen 12-16 week-old New Zealand male rabbits,weight range from 2.5kg to 3kg,without any ocular disease by Slit lamp were Provided from the animal experimental center of Hebei Medical University.They were all first adaptive feeded In the clean animal room for 7 days,contacted with the experimental staff to adapt to the environment,while were trainned to accept for local eye drops,and to adapt to surface anesthesia underwent intraocular pressure measurement.In Feeding room,we controlled the temperature at 21-24 ℃,relative humidity at 55%-65%.The room was keep ventilated dry,quiet.12 hours alternating sunlight,daily diet,free water for animals.Fifteen rabbits were randomly divided into normal control group(n = 5)and experimental group(n = 10).Schiotz tonometer was used to measure the intraocular pressure with Captopril eye drops surface anesthesia.Each eye was measured twice and then took the average.,Tobramycin eye drops was applicated to prevent infection after the measurement.The IOP of each rabbit was measured at 8: 00,10: 00,12: 00 daily for 3 days before administration,and the mean value was obtained.On the fourth day,the experimental group was given by 0.5% dexamethasone with subconjunctiva injection 5mg(1ml)at a fixed time(8:00)and place every day in the rabbit left eye,and then use the Tobramycin eye drops and ointment to prevent infection.At the next day the left eye was administration with 0.1% dexamethasone eye drops 4 times a day for consecutive 7 weeks and the the same method was used with sterile normal saline solution to the control group.monitored IOP every 3 days.The successful criteria of model eyes was defined as rising of IOP to≥24 mmHg for over 1 week.After 7-week administration,the rabbits were sacrificed by excessive anesthesia.Use a sharp surgical blade and scissors to cut the eyeballs along the equator,discard the behind half and carefully remove the lens.The anterior segment include the cornea,iris,ciliary body,atrial angle,and anterior sclera were cut open.Half of them were fixed in 10% neutral buffered formalin for 48 h to prepare a paraffin specimen for HE staining and immunohistochemical staining;the other half of the specimens reserved the TM and ciliary body tissue only and put into the small liquid nitrogen bottles,stored in the refrigerator at-80 ℃ in order to prepare for Reverse transcription-PCR(RT-PCR).Total RNA was extracted from cryopreserved ciliary body tissue homogenate and detected by spectrophotometer,then cDNA was synthesized by reverse transcription,and6% of the amplified product of each sample was mixed with 1% agarose gel electrophoresis containing GV nucleic acid dye,Labeled with DNA Marker as standard fragment,electrophoretic observation by UV transmittance,and took photals.Use the Quantity One to practice the image analysis.The result is expressed as the ratio of the absorbance of the two electrophoresis strip.Statistical analysis was performed by SPSS21 statistical software(IBM,USA).The data of the experimental test data were positively distributed by the Shapiro-Wilk test.The difference of intraocular pressure between the experimental group and the control group was compared with that of the two groups.The relative expression of llβ-HSD1 gene in rabbit ciliary body was compared with the independent sample t test.P <0.05 for the difference was statistically significant.Results: The average IOP was 18.97±2.92 mmHg.The baseline of the experimental group was 19.42±2.10 mmHg and the control group was18.53±2.99mmHg(P> 0.05).In the experimental group,the IOP was elevated26.07±2.17 mmHg at the third week,and reached the peak26.77±4.37 at the fourth week.There was a significant statistical difference for the IOP between the experimental group and the control group from the third week.There was no significant difference in IOP of control group during the 7weeks(P> 0.05).The IOP increased by 90% in the experimental group.Compared with the normal control group,the TM cells with the HE staining showed nuclear staining darker,the boundary was blurred,the extracellular matrix was obviously increased,and the gap among TM is reduced.There were no obvious morphological changes in Ciliary body between the two groups of rabbit eyes.1lβ-HSD1 protein was positively expressed in the ciliary body of rabbits in both the experimental group and the control group.The positively expression of 1lβ-HSD1 protein was in non-pigmented epithelial cells of ciliary,wile the pigment epithelium and the stroma layer showed negative expression,and the expression was weaker in the control group compared with the experimental group.RT-PCR showed that the expression level of llβ-HSD1 mRNA in the ciliary body of the experimental group was higher than that in the control group.Quantitative analysis of Quantity One software showed that the average expressional value of llβ-HSD1 in rabbits was 0.86±0.07 in the experimental group and 0.35±0.06 in the control group,and the difference between the two groups was statistically significant(P﹤0.05).Conclusion:1 Subconjunctival injection combined with ocular surface dexamethasone can successfully induce the increase of IOP in New Zealand white rabbits.2 Compared with normal control group,the TM cells with the HE staining showed nuclear staining darker,the boundary was blurred,the trabecular meshwork was disturbed,the extracellular matrix was increased,and the gap between the trabecular beam was narrowed.3 The expression of llβ-HSD1 in ciliary body non-pigment epithelium was higher in the experimental group than that in the control group,suggesting that the increase of exogenous glucocorticoid could stimulate the expression of llβ-HSD1,and then promote the production of endogenous glucocorticoids so that elevate the IOP. |
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