| Part I Ultrasonic measurement of the rabbit ophthalmic artery and optic nerve:A reproducibility and repeatability Study ObjectiveTo evaluate the repeatability and reproducibility of color Doppler flow imaging(CDFI)measurement of hemodynamic parameters of ophthalmic artery(OA)and transorbital ultrasound(TOUS)measurement of optic nerve diameter(OND)in the rabbits,and provided references for further research.Methods 1.The reliability of CDFI measurement of OA hemodynamic parameters: two independent operators examined the 31 rabbits(62 eyes)in the following sequence: operator 1?operator 2?operator 1.Five Doppler velocimetry indices,the peak systolic velocity(PSV),end-diastolic velocity(EDV),resistance index(RI),pulsatility index(PI)and the ratio of peak systolic velocity to end-diastolic velocity(S/D)were measured.The examination interval was less than 10 minutes.2.The reliability of TOUS measurement of OND: two independent operators took the examination in the following turn: operator 1?operator 2?operator 1.OND of the rabbit was measured 3mm behind the optic disk.3.Differences between the two measurements made by operator 1 comprised the ?repeatability? score,while differences between the first values obtained by operator 1 and the values of operator 2 comprised the ?reproducibility? score.Results1.All of the OA and optic nerve in the 31 rabbits were well imaged by CDFI and TOUS.The Doppler spectrum of rabbit ophthalmic artery is similar to that of humans,showing a “three peak and two valley” shape.However,the late systolic notch is not as obvious as that of human beings.The optic nerve of rabbit located behind the eyeball as a small hypoechoic strip.2.All ICC values of the five OA Doppler blood flow parameters measured by the same examiners were more than 0.75,which implied high reproducibility.The results of the correlation analysis showed that the values of the blood flow parameters of the different examiners were all significantly correlated,all P<0.001,and the measurement consistency was high.The Bland-Altman plots further confirmed that the Doppler blood flow parameters measurement agreement of the rabbit OA were high by different operators.3.The OND measured by operator 1 was 1.19±0.12 mm and 1.17±0.11 mm separately.AND the OND measured by operator 2 was 1.17±0.11 mm.No statistically difference was noticed among the three measurements(P=0.776).The ICC value of OND measured by the same operator was 0.973,and the OND values measured by different operators were significantly correlated(r=0.905,P<0.001).Bland-Altman scatter plot also showed that TOUS OND measurement was highly repeatable.ConclusionThe CDFI measurement of rabbit OA and TOUS measurement of rabbit OND is feasible and reliable.The ultrasonic technology can be widely used in the future research.The results of the present study provide a reliable reference for the future study.Part II A study on the correlation between hormones,tear secretion function and retrobulbar hemodynamics ObjectiveTo explore the role of retrobulbar hemodynamics in the pathogenesis of dry eye(DE)by analyzing the relationship between human sex hormone levels,tear secretion and retrobulbar hemodynamics.Methods 1.148 healthy volunteers were enrolled between September 2016 and October 2017.All of the subjects were divided into menopause group(MP)and non-climacteric group(NMP)according to the time of menopause.2.The height,weight,blood pressure,heart rate,Shirmer I test(SIt)and tear film breakup time(BUT)were measured and recorded before the ultrasonic examination.3ml blood was extracted fasting and stored in a-80°C refrigerator after high speed centrifugation.Serum estradiol(E2)and testosterone(T)concentration were determined by electrochemiluminescence immunoassay.3.The hemodynamic parameters of OA,CRA,and SPCA in each subject were measured,including PSV,EDV,RI,PI,and S/D.The mean value of binocular measurements was taken to statistical analysis.4.Independent sample t test was used to compare the age,height,weight,blood pressure,heart rate,SIt,BUT,E2 and T difference between the two groups.Pearson correlation was used to analysis the relationship between age,E2,T,BUT,SIt and hemodynamic parameters.Multiple linear regression was used to screen factors that influence the SIt and BUT separately.Results1.A total of 148 subjects were included in the present study,including 75 persons in group NMP and 73 in group MP,with an average age of 36.6±9.0 years and 62.5±10.2 years respectively.No statistically difference was noticed of the height,weight,heart rate,systolic blood pressure(SBP)and diastolic blood pressure(DBP)between the two groups(all P > 0.05).2.The value of E2,T,SIt and BUT was significantly different between the two groups(all P<0.05).The values of E2,T,SIt and BUT in group NMP were significantly higher than that of group MP.The PSV,EDV,RI and S/D values except for PI in the OA,SPCA and CRA were all significantly different(all P<0.05).The OA velocity in the MP group was lower than that of NMP group,but the resistant indices were higher.The hemodynamic parameters of CRA were all significantly different except for PSV and PI.The value of EDV in MP group was lower than NMP group,while the RI and S/D value was higher than the NMP group.All of the hemodynamic parameters of SPCA showed significantly different between the two groups except PI(all P<0.05).The values of PSV and EDV were much lower in the MP group,while the RI and S/D was higher.3.The Pearson correlation results showed that age correlated negatively with E2,T,SIt,BUT,OA-PSV,OA-EDV,CRA-EDV,SPCA-PSV and SPCA-EDV but correlated positively with OA-RI,OA-S/D,CRA-RI,CRA-S/D,SPCA-RI,SPCA-S/D(all P<0.05).No statistical correlation was found between age and the other indices.4.The Pearson correlation results showed that serum E2 value correlated negatively with age,OA-RI,OA-S/D,CRA-RI,CRA-S/D,SPCA-RI and SPCA-S/D but correlated positively with SIt,BUT,OA-PSV,OA-EDV,CRA-EDV,SPCA-PSV and SPCA-EDV(all P < 0.05).No statistically correlation was found between E2 and the other indices.5.The Pearson correlation results showed that serum T level correlated negatively with age,OA-RI,OA-PI,OA-S/D and SPCA-PI but correlated positively with SIt,BUT,OA-PSV,OA-EDV,SPCA-PSV and SPCA-EDV(all P<0.05).No statistically correlation was found between T and the other indices.6.The Pearson correlation results showed that SIt correlated negatively with age,OA-RI,OA-S/D,CRA-RI,CRA-S/D and SPCA-RI but correlated positively with E2,T,OA-PSV,OA-EDV,CRA-PSV,CRA-EDV,SPCA-PSV and SPCA-EDV(all P<0.05).No statistically correlation was found between SIt and the other indices.7.The Pearson correlation results showed that BUT correlated negatively with age,OA-RI,OA-S/D,CRA-RI,CRA-S/D and SPCA-RI but correlated positively with E2,T,OA-PSV,OA-EDV,CRA-PSV,CRA-EDV,SPCA-PSV and SPCA-EDV(all P<0.05).No statistically correlation was found between BUT and the other indices.8.Multiple linear regression analysis of SIt and related factors showed that SIt was positively correlated with serum E2 level and OA-PSV,and negatively correlated with age(R2=0.695,P<0.05),and the fitting model was statistically significant on the whole(F=109.392,P < 0.001).The regression equation was ?=6.350-0.118X1+0.225X2+0.038X3(? represents SIt,X1 represents age,X2 represents E2,X3 represents OA-PSV).The estimated value of the standard partial regression coefficient showed that the effect of E2 on SIT was greater than the effect of age and OA-PSV.9.Multiple linear regression analysis of BUT and related factors showed that BUT was positively correlated with serum E2 level and OA-PSV,and negatively correlated with age(R2=0.712,P<0.05).The fitting model was statistically significant on the whole(F=118.641,P < 0.001).The regression equation was ?=6.427-0.122X1+0.223X2+0.037X3(? represents BUT,X1 represents age,X2 represents E2,X3 represents OA-PSV).The estimated value of the standard partial regression coefficient showed that the effect of E2 on BUT was greater than the effect of age and OA-PSV.Conclusion1.The level of sex hormone is closely related to the retrobulbar hemodynamics.2.Aging,reduced E2 and OA-PSV decrease are independent factors affecting SIt and BUT,in which the E2 levels influent the most.3.OA hemodynamics abnormal may play a role in the pathogenesis of DE.Part III Effects of HRT on tear secretion,serum NO and retrobulbar hemodynamics in ovariectomized rabbitsObjectiveTo study the effect of hormone replacement therapy(HRT)on tear secretion,serum nitric oxide(NO)and retrobulbar hemodynamics,and further explore the role of hemodynamics change in the pathogenesis of DE.Methods36 healthy adult female rabbits were randomly divided into four groups: the sham operation group(Group F),experimental control group(Group C),estrogen treatment group(Group E)and androgen treatment group(Group T),9 rabbits for each group.Group F only underwent midline laparotomy,and then sutured the wound with the ovaries being found out but not resected.The remaining three groups were treated with bilateral ovariectomy.After 7 days ovariectomy,Group E was treated with 17-?estradiol with a dose of 0.5mg/kg/d for 28 days.Group T was treated with dihydrotestosterone with a dose of 4mg/kg/d for 28 days.Rabbits in group C were given no treatment.2.The values of SIT,BUT,corneal fluorescein staining and OA hemodynamic parameters(PSV,EDV,RI,PI and S/D)were obtained before operation(D0),7 days after operation(D7),and 28 days after hormone treatment(D28).3ml of fasting blood was extracted from each subject and preserved in-80°c refrigerator after high speed concentrated.Griess method was used to detect the concentration of NO.3.One-way ANOVA analysis was used to compare the difference of each value at different time points in different groups.Variation at the three time points in the same group was compared by repeated measurement ANOVA.Results1.One rabbit died in both group F and group T for anaesthetic thunderbolt.And the number of rabbit in each group was 8 in group F,9 in group C,9 in group E and 8 in group T,with a total number of 34.2.Results of corneal fluorescein staining.The corneal fluorescein staining of each rabbit before surgery in every group was negative.And the corneal fluorescein staining of group F in D7 and D28 was both negative.Mild corneal fluorescein staining was observed in group C,group E and group T at D7.Result of one-way ANOVA analysis showed that no significant difference was found among the three groups(F=0.106,P=0.900).The staining in group C and group E was more serious at D28(with t value of 3.790 and 5.352,and P values of 0.005 and 0.001).The cornel staining integral of group T was significant lower at D28 than D7(t=13.899,p<0.001).3.Results of SIt.The value of SIt in each group showed no statistical difference at D0 but obvious difference was noticed at D7.Multiple comparison results showed that there was no difference among group C,group F and group T,but significantly different from group F.SIt difference of four groups was obvious(F=8.611,P=<0.001)in D28.The SIt value of group T increased and showed no difference with group F.The values of group C and E were lower than group T and F.No significant difference was observed of SIt values in group F at three time points(F=0.016,P=0.984).However,there was obvious difference of SIt values in group C at three time points(F=66.446,P<0.001).The SIt value decreased as time went on.Significant difference of SIt in group E at three time points was observed(F=39.198,P<0.001),showing declined trend as time went by.Obvious difference of SIt in group C at different time points was also observed(F=24.946,P<0.001),with the value of D28 equaled to D0 and higher than D7.4.Results of BUT.The value of BUT in each group showed no statistical difference at D0 but obvious difference was noticed at D7.Multiple comparison results showed that there was no difference among group C,group F and group T,but significantly lower than group F.BUT difference of four groups was obvious(F=8.611,P<0.001)in D28,with the value of group F equaled to group T and higher than group C and E.NO significant difference was observed in the latter two groups.No significant difference was observed of BUT in group F at three time points(F=0.476,P=0.643).However,obvious difference of BUT was seen in group C at three time points(F=56.283,P<0.001),with the value of D28 lower than D7 and D0.Significant difference of BUT in group E at three time points was observed(F=356.225,P<0.001),with the value of D28 lower than D7 and D0.Obvious difference of BUT in group T at different time points was also observed(F=40.140,P<0.001),with the value of D28 equaled to D0 but higher than D7.5.Results of serum NO level.The value of NO of each group showed no statistical difference at D0(F=0.051,P=0.984)but obvious difference was noticed at D7(F=9.815,P<0.001).Multiple comparison results showed that there was no difference among group C,group F and group T,but significantly different from group F,with the value group C equaled to group E and T but lower than group F.NO level difference of four groups was obvious(F=11.984,P<0.001)in D28,with the value of group F equaled to group E but higher than group T and C.No significant difference was observed of NO level in group F atthree time points(F=0.324,P=0.729).However,obvious difference of NO concentration was seen in group C at three time points(F=64.697,P<0.001),with the value of D28 equaled to D7 but lower than D0.Significant difference of NO concentration in group E at three time points was observed(F=24.265,P<0.001),with the value of D28 equaled to D0 while lower than D7.Obvious difference of NO level in group T at different time points was observed(F=20.690,P<0.001),with the value of D0 higher than D28 and D7?6.Compared results of OA hemodynamic indices.There was no significant difference in the hemodynamic parameters of the OA in each group before ovariectomy(all P>0.05).All parameters except S/D were significant different at D7(all P<0.05).Both values of PSV and EDV in group C,E and T showed no difference but lower than group F,while RI and PI in group C,E and T higher than group F.All parameters except S/D were significant different at D28(all P<0.05),with values of both PSV and EDV in group F equaled to group T but higher than group C and equaled to group E.RI of group F equaled to group T but lower than group C and E.PI of group F was lower than group C and E but higher than group T.No significant difference was observed of all parameters in group F at three time points(all P>0.05).All parameters except S/D were significant different in group C at three time points(all P<0.05),with both PSV and EDV of D0 higher than D7 but equaled to D28,while both RI and PI of D0 lower than D7 but equaled to D28.All parameters except S/D were significant different in group E at three time points(all P<0.05),with both PSV and EDV of D0 higher than D7 but equaled to D28,while both RI and PI of lower than D7 but equaled to D28.All parameters in group T were significant at three time points(all P<0.05),with both PSV and EDV of D0 equaled to D28 but higher than D7,while all RI,PI and S/D of D0 equaled to D28 but lowerthan D7.The above results suggested that ovariectomy might reduce blood flow velocity and increase blood vessel resistance of OA in female rabbits.Supplementation of estrogen might further reduce the blood flow velocity of OA and increase blood vessel resistance,while the effect of androgen supplement was opposite.Conclusion1.Ovariectomy and HRT have different effects on serum NO,tear secretion and OA hemodynamic parameters,indicating that sex hormone plays an important role in maintaining the balance of tear secretion and retrobulbar hemodynamics.2.NO is an important factor in regulating the retrobulbar hemodynamics by sex hormones.3.There is a certain correlation between hemodynamic changes and the occurrence and development of DE. |
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