| ObjectiveTo study the effects of long-term application of prostaglandins on conjunctiva l thickness and conjunctival lymphatic vessels and the concentration level of PGE2 in aqueous humor following acupuncture in the rabbit eyes.Methods1.Twenty four healthy New Zealand White rabbits were randomly divided into latanoprost group,carteolol control group and control group with each group having 8 rabbits.The conjunctival thickness was measured by optical coherence tomography and recorded at baseline and two months after administration of latanoprost eye drops(once a day)and carteolol eye drops(once a day)and the conjunctival thickness was compared between the two time points in both groups.2.Western Blot,RT-PCR and immunohistochemistry were used to analyze and detect the changes in the level of Matrix Metal oproteinases-1(MMP-1),Matrix Metal oproteinases-3(MMP-3)protein and m RNA expression in the conjunctiva l tissues.3.Western Blot,RT-PCR and immunofluorescence were also used to investiga te the changes in the levels of 5’-nucleotidase(5’-Nase)in conjunctival lymphatic vessels and Alkaline phosphatase(ALPase)in conjunctival capillaries after long-term latanoprost eye drops administration;5’-nucleotidase-Alkaline phosphatase histochemical staining was used to identify conjunctival lymphatic vessels and conjunctival capillaries in rabbits and to observe the distribution of conjunctiva l lymphatic vessels in conjunctival tissues.4.For the acupuncture study,acupunctures were set at bilateral Jingming,Taiyang and Tongziliao acupoints with the needle being retained at each point for 20 minutes.The aqueous humor was extracted at 1 hour,4 hours and 8 hours after acupuncture,respectively.The changes in the levels of endogenous PGE2 were explored by ELISA at the time-points mentioned above.Results1.There was a significant difference in the conjunctival thickness of the rabbits in the latanoprost group between the baseline(178.88 ± 12.03 μm)and post-experiment 2 months(124.19 ± 11.29 μm),(F=309.094,P<0.001).while no significant differe nce was found in the carteolol group between the baseline(184.94 ± 11.85 μm)and postexperiment 2 months(183.31 ± 8.7 μm).(F=0.195,P=0.662).2.The expression level of MMP-1 in the conjunctival tissue in the latanoprost group(1.04 ± 0.074)was significantly higher than in the control group(0.86 ± 0.030)by Western Blot.(F=26.980,P<0.001);The m RNA expression level of MMP-1 in the conjunctival tissue in the latanoprost group(5.55 ± 1.615)was significantly higher than in the control group(1.00 ± 0.008)by RT-PCR.(F=38.131,P<0.001);3.The expression level of 5’-Nase in the conjunctival tissue in the latanoprost group(0.99 ± 0.095)was significantly higher than in the control group(0.83 ± 0.011)by Western Blot.(F=231.175,P<0.001);The m RNA expression level of 5’-Nase in the conjunctival tissue in the latanoprost group(2.64 ± 0.398)was significantly higher than in the control group(1.00 ± 0.009)by RT-PCR.(F=84.115,P<0.001);The intensity of the 5’-Nase in the latanoprost group was higher than control group by fluoresce nt staining.There was no difference in the protein expression level of ALPase between the latanoprost group and the control group(F=1.247,P=0.788);The conjunctiva l lymphatics were differentiated from the conjunctival capillary by the enzymat ic histochemistry staining.4.The expression level of endogenous prostaglandins in the aqueous humor was up-regulated after acupuncture,which peaked at 4 hours after acupuncture(F=56.561,P<0.001).Conclusions1.The long-term topical use of prostaglandins can significantly thin the overall conjunctiva.The increased protein expression of MMP-1 in conjunctival tissue may be one of the causes involved in thinning the conjunctiva.2.The long-term topical application of prostaglandins could upregulate the expression of 5’-nucleotidase.This may argue that exogenous prostaglandins can proliferate the conjunctival lymphatic vessels.3.Acupuncture at specific acupoints around the eyes can significantly upregula te the expression of endogenous PGE2 in aqueous humor,which may be the mechanis m by which the acupuncture has been used to treat glaucoma. |
