Effects Of Glucocorticoids On The Function Of Trabecular Meshwork And Its Regulation

Glucocorticoid(GC)is a hormone with multiple physiological functions and a widely used drug in clinical practice.With the development of the aging of the society,the need for glucocorticoid treatment is increasing.One of the problems with glucocorticoids is that different patients have different responses and certain side effects.In ophthalmology,glucocorticoids are also required for the treatment of a number of ocular diseases.In the eyes,long-term application of glucocorticoid therapy can cause cataracts,elevated intraocular pressure and even glaucoma,the latter can cause damage to the optic nerve,causing irreversible damage to the visual function and bringing great impact to patients.Trabecular Meshwork and Schlemm's Canal(TM-SC)is the main route for drainage of aqueous humor.TM-SC plays an important role in regulating drainage of aqueous humor and maintaining stable intraocular pressure.Glucocorticoid can affect the function of trabecular meshwork and increase the stiffness of trabecular meshwork,leading to the increase of resistance to external drainage of aqueous humor and the increase of intraocular pressure.Therefore,it is of great significance to explore the mechanism of glucocorticoid induced ocular hypertension and the body's different sensitivity to glucocorticoid in detail.The function of glucocorticoid is mediated by its receptor(GR),which can be divided into different subtypes.GRaand GR?are the two most studied subtypes,and GR?is generally considered to be a natural inhibitor of GR?-mediated glucocorticoid functions.The expression difference of the two receptor subtypes in different tissues and cells may lead to different sensitivity to glucocorticoids.Previous studies on systemic diseases have found that the cytokines such as IL-1?can lead to glucocorticoid resistance(GCR)through the changes of GRs.In ophthalmology,most studies about IL-1?functions on IOP were about the matrix metalloproteinases(MMP).Those studies showed that IL-1? could change the expression of MMP,which can influence the configuration of the extracellular matrix and result in decreased aqueous outflow resistance.However,the effect of IL-1?on ocular glucocorticoid receptors has not been studied.In the first part of this study,dexamethasone(DEX)was used to treat the human trabecular meshwork cells(HTMC).The effects of DEX on the expression of GRaand Gr?and its influence on the expression of trabecular meshwork sclerosis related indicators were observed.The function of IL-1?was also evaluated.In the second part,the DEX induced ocular hypertension model was made in rats.The changes of intraocular pressure and the expression of GR isoforms and related factors in the trabecular meshwork of rats were measured.In the third part,a new method was tested in evaluating the function of TM.The images of TM-SC area was segmented and measured to find the relationship between morphology and the intraocular pressure changes,which could serve as another indicator of trabecular meshwork function.Part ? Effects of glucocorticoids on human trabecular meshwork cell function and its regulationObjective:To investigate the effect of glucocorticoids on the expression of GR subtypes in human trabecular meshwork cells and the regulation effect of IL-1?on the expression of GR subtypes in human trabecular meshwork cells.Methods:1.Human trabecular meshwork cells were treated with different concentrations of IL-1?(0ng/mL,10ng/mL,20ng/mL,30ng/mL,40ng/mL)and different time duration(0h,6h,24h,48h).The expression of GRa and GR?mRNA on human trabecular meshwork cells was detected by RT-PCR.2.The concentration and time duration with greatest changes of GRa/Gr? ratio for IL-1? treatment was selected according to step 1 results and combined with DEX treatment.RT-PCR and Western blot were used to observe the regulatory effects of IL-1? pretreatment on mRNA and protein changes of GRa and GR? in human trabecular meshwork cells induced by DEX and DEX combined with IL-1 ?.3.Elisa was used to further detect the expression of MYOC and FN in human trabecular meshwork induced by DEX and the regulatory effect of IL-1? on the expression.Results:1.Effects of different concentrations of IL-1? on the expression of GR in human trabecular meshwork cells:The mRNA expression of GRs in human trabecular meshwork cells increased first and then decreased with the increase of concentration after treatment with IL-1? at different concentrations(10ng/mL,20ng/mL,30ng/mL,40ng/mL)for 24h,and the increase degree of GR?was more obvious.The greatest increase was found when using 20ng/mL IL-1?.There was no statistically significant difference in GRamRNA expression between the 10ng/mL IL-1?treatment group and the control group(Ong/mL)(P>0.05).There was statistically significant difference in GRamRNA expression between the 20ng/mL group and the 30ng/mL group(P<0.05),and there was statistically significant difference in GR?mRNA expression among the other treatment groups at different concentrations(P<0.01).There was no statistically significant difference in GR?mRNA expression between the 10ng/mLIL-1?treatment group and the control group(Ong/mL)(P>0.05),while there was statistically significant difference in GR?mRNA expression among the other treatment groups at different concentrations(P<0.01).2.Effects of different time duration of IL-1? on glucocorticoid receptor expression in human trabecular meshwork cells:The mRNA expression of GRs in human trabecular meshwork cells increased first and then decreased with time after treatment with 20ng/mgIL-1? for different time duration(6h,24h,48h).The expression of GRs peaked at 24h,and the increase degree of GR? was greater.There was no statistically significant difference in GRamRNA expression between the 48h group and the blank control group(P>0.05).There were statistically significant differences in GRamRNA expression among the other groups at different treatment times(P<0.01).There was no statistically significant difference in GRPmRNA expression between the 6h group,48h group and the blank control group(P>0.05),while there was statistically significant difference in GR?mRNA expression among the other treatment groups at different times(P<0.05).3.Effects of IL-1? on DEX induced changes in glucocorticoid receptor expression in human trabecular meshwork cells:The expression of GRs in human trabecular meshwork was decreased after treatment with 100nM DEX for 48h,but this was changed by 20ng/mL IL-1? pretreatment for24h.The influence on GR?was more obvious.There was no significant difference in GRamRNA expression between the blank control group and the IL-1?pretreatment group(P>0.05),while there was significant difference in GRamRNA expression between the blank control group,IL-1?pretreatment group and the DEX treatment group(P<0.01).There were statistically significant differences in GR?mRNA expression between the blank control group and the IL-1?pretreated group(P<0.05),and there were statistically significant differences in GR?mRNA expression between the blank control group,the IL-1?pretreated group and DEX group(P<0.01).The influences on GR?and GR?protein expression were consistent with mRNA expressions among groups.4.Influence of IL-1?on DEX induced changes in human trabecular meshwork cell sclerosis indexThe mRNA and protein expressions of MYOC and FN in human trabecular meshwork cells were increased after treatment with 100nM DEX for 48h and decreased by pretreatment with IL-1?,which was consistent with the trend of increased expression of GR?in trabecular meshwork cells caused by IL-1?.There were no statistically significant differences in the mRNA expressions of MYOC and FN between the blank control group and the IL-1?combined with DEX group(P>0.05).There were statistically significant differences in the mRNA expressions of MYOC and FN between the DEX group and the blank control group,and between the DEX group and the combined group(P<0.01).The influence of IL-1?on MYOC and FN protein expression in each group was consistent with the influence trend of mRNA expression.Conclusions:1.DEX treatment can affect the expression of GRa and GR? in trabecular meshwork cells.2.IL-1? pretreatment can influence the changes of GRa and GR? induced by DEX in trabecular meshwork cells.3.The expression of GR?/? in trabecular meshwork cells was correlated with the degree of DEX induced trabecular meshwork stiffening.Part ? Effects of glucocorticoids on the function of rat trabecular meshwork and its regulationObjective:To investigate the effect of glucocorticoids on the IOP and expression of GR isoforms in rats and the regulation effect of IL-1?.Methods:1.Animals:Forty-five rats were randomly divided into three groups,with each rat's right eye as the experimental eye and its left eye as the control eye.Group A was blank control group without any treatment.Group B was treated with DEX and group C was treated with DEX combined with IL-1?.2.Animal model preparation:The basic intraocular pressure was determined after chloral hydrate abdominal anesthesia and benoxinate hydrochloride eye drop.Rats in group B and Group C received drug injection.After anesthesia,the eye conjunctival sac was routinely disinfected and rinsed with normal saline.The drugs or 0.1 mL of normal saline were injected subconjunctivally with a 29G syringe.After the injection,one drop of 0.3%levofloxacin eye drop was applied to prevent infection.In group B,0.5%dexamethasone sodium phosphate was injected twice a week in the right eye while normal saline in the left eye.In group C,50ng of IL-1?was applied to the right eye on the first day of treatment and the followed treatment was the same as that in group B from the next day.After the first injection,animals in group B and Group C received DEX(experimental eye)or normal saline(control eye)eye drops three times a day.The intraocular pressure was measured under anesthesia before each injection.3.Indicators:Conjunctival congestion,anterior chamber inflammatory cells,pus in anterior chamber,iris congestion and pupil diameter were observed under slit lamp at 3 days,2 weeks and 1 month after injection.The inflammation scores was evaluated.The rats were sacrificed and enucleated at 3 days,2 weeks and 1 month.The eyeballs were dissected and stored in-80? for RT-PCR or prepared for paraffin sections for HE staining and immunohistochemical detection.Results:1.IOP results:The IOP in the DEX treatment group increased gradually,and the IOP increased from the baseline level of 6mmHg to 12mmHg one month after treatment.The IOP of the control eye fluctuated around 6mmHg.IOP of the DEX combined with IL-1?group decreased on the 3rd day,and was higher than that of the control group on the 10th day,and then gradually increased.2.Slit lamp examination:No obvious abnormalities were found in the DEX treatment group,and the inflammation score was 0.In the DEX+IL-1?group,the conjunctiva and iris were congested at 3 days with slightly decreased pupil diameter.3.HE staining:Ciliary cysts appeared in some experimental eyes in the DEX group at 1 month,and no significant difference was found in the other experimental eyes compared with the control eyes and the blank control eyes.No obvious inflammatory cell infiltration was observed in the combined IL-1?group.Only the number of blood vessels in the iris and ciliary body was higher than that in the control eye at 3 days after injection.4.RT-PCR results:(1)GRa expression:The mRNA level of GRa in DEX group decreased gradually.Compared with the control group,there was no significant difference in DEX treatment for 3 days(P>0.05),and there was significant difference in DEX treatment for 2 weeks and 1 month(P<0.05).Pairwise comparisons between the 3-day group,the 2-week group and the 1-month group were statistically significant(P<0.05).After IL-1?pretreatment,GR mRNA expression increased at 3 days and decreased at 1 month.Compared with the control group,there was no statistically significant difference between the 2-week group(P>0.05),and there was statistically significant difference between the 3-day group and the 1-month group(P<0.05).Pairwise comparisons between the 3-day group,the 2-week group and the 1-month group were statistically significant(P<0.05).There were statistically significant differences between the DEX group and the DEX combined IL-1?treatment group at 3 days and 2 weeks(P<0.05),but no statistically significant difference between 1-month groups(P>0.05).(2)GR? expression:The expression of GR? was detected in the liver and spleen of rats,but the GR? level was much lower than that of GR?.No expression of GR? was detected in the trabecular meshwork of the eyeball.(3)MYOC and FN expression:After DEX treatment,the expression of MYOC and FN mRNA increased gradually,while that of IL-1? pretreated for 3 days decreased and then increased gradually.Compared with the control group,there was no significant difference in DEX treatment for 3 days(P>0.05),and there was significant difference in DEX treatment for 2 weeks and 1 month(P<0.05).Pairwise comparisons between the 3-day group,the 2-week group and the 1-month group were statistically significant(P<0.05).After IL-1? pretreatment,MYOC and FN mRNA expression was decreased in the 3-day group and increased in the 1-month group.Compared with the control group,there were no statistically significant differences in the 2-week group(P>0.05),and there were statistically significant differences between the 3-day group and the 1-month group(P<0.05).Pairwise comparisons between the 3-day group,the 2-week group and the 1-month group were statistically significant(P<0.05).There were statistically significant differences between the DEX group and the DEX combined IL-1?treatment group at 3 days and 2 weeks(P<0.05),but no statistically significant difference between 1-month groups(P>0.05).5.IHC results:(1)Expression of GR?:The expression of Gr?protein was similar to the mRNA level?(2)Expression of GR?:In the blank control group,the corneal epithelium,trabecular meshwork,ciliary epithelium and lens epithelium were all stained with brownish yellow granules.The expression of Gr?showed no difference between different groups at different time points.(3)Expression of MYOC and FN:After treatment with DEX,MYOC and FNexpression increased.There was no difference between the 3-day group and the control group(P>0.05),and the difference between the other groups and the control group was statistically significant(P<0.05).After pretreatment with IL-1?,the expressions were decreased in the 3-day group and increased in the 2-week group and the 1-month group,compared with control eyes(P<0.05).There was no significant difference between the DEX group and the DEX combined IL-1?group at 1 month(P>0.05),and there were significant differences between the DEX group and the DEX combined IL-1?group at other time points(P<0.05).Conclusions:1.DEX eye drops combined with subconjunctival injection of DEX can increase the IOP in rats.2.IL-1? can reduce the IOP increase caused by DEX.3.MYOC and FN can be used to evaluate the stiffness of trabecular meshwork.4.The effect of IL-1? on IOP may be independent of the expression of GR isoformsPart ? Research on new technology for trabecular meshwork function evaluation in vivoObjective:To evaluate the different segmentation methods and quantitative analysis of SC-TM in UBM images.Methods:1.26 healthy subjects were recruited and evaluated to meet the inclusion criteria.After the baseline IOP was measured,the subject began to play the trumpet and kept it as long as possible.The UBM images of one eye was obtained while the IOP of the other eye was measured at different time points:10s after the beginning of trumpet blowing,immediately after blowing and 10s after blowing stopped.All images were collected at the inferior part of the anterior chamber angle.2.Images were screened according to the image inclusion criteria.ImageJ software and K-means,FCM and Level-Set segmentation algorithms were respectively used to segment the UBM images obtained.The TM-SC region was quantitatively analyzed based on the segmentation results.The relative error between the three algorithms and the results of ImageJ analysis was used to quantify the accuracy of the geometric measurement of the three segmentation algorithms.Paired T-test was used to analyze the differences among the geometric measurement results of K-means,FCM and Level-Set segmentation with that of ImageJ.ICC was applied to quantify the credibility of K-means,FCM and Level-Set segmentation algorithms.3.Based on the comparison results of different segmentation methods,the optimal segmentation method was selected to conduct quantitative analysis of all the images.The correlation between the geometric measurement results and the corresponding intraocular pressure measurement values was quantified by Pearson correlation coefficient.Results:1.General conditions:a total of 104 UBM images were obtained from 26 subjects,among which 84 images met the inclusion criteria.The mean IOP before and 10s after blowing the trumpet was 17.5mmHg and 28.8mmHg,respectively.2.Image segmentation:Four segmentation methods were used to segment UBM images in different states of the same subject respectively.It showed that the Level set method could clearly identify the boundary between TM and SC regions,with no noise influence and small difference from the real value.It was applicable to different IOP states.3.Comparing the segmentation results of the three methods with those of ImageJ:The Level-set method has better accuracy,reliability and consistency than the other two segmentation methods compared with ImageJ.There were no statistically significant differences between the measurement data obtained by the level-set method and ImageJ(p=0.663,p=0.071,p=0.755,and p=0.117 for SC area,SC perimeter,SC length and TM width,respectively).There were no statistically significant differences in the SC area and SC perimeter measurements between the K-means method and by ImageJ(p=0.103 and p=0.901 for SC area and SC perimeter respectively),while the differences for the SC length and TM width between the two methods were statistically significant(p<0.001 for SC length and TM width).There was no statistically significant difference between the SC area measured by the FCM method and the corresponding result measured by ImageJ(p=0.662),while the differences in the SC perimeter,SC length and TM width between the two methods were statistically significant(p=0.032,p<0.001,and p<0.001 for SC perimeter,SC length,and TM width,respectively).The relative errors of the level-set method are less than 0.08,whereas the other two methods have relatively large relative errors.The ICC values of the level-set method are 0.91,0.95,0.9,and 0.57,respectively,whereas the corresponding ICC values of the other two methods are less than 0.4.4.The relationship between the quantified segmentation results and IOP by the Level set method:Pearson correlation coefficients between IOP and SC area,SC perimeter,SC length and TM width were-0.91,-0.72,-0.66 and-0.61,respectively(P<0.01).Conclusions:1.The level-set method showed better accuracy than the other two methods.Compared with manual methods,it can achieve similar precision,better repeatability,and greater efficiency.Therefore,the level-set method can be used for reliable UBM image segmentation.2.The SC area segmented by the level-set method has a good negative correlation with IOP,which may be used as a new index for the function evaluation of trabecular network.