Lowing-intraocular pressure and improving aqueous humor dynamic effects of qingguangan drug granules in spontaneous glaucoma mice / 中华实验眼科杂志

Background Researches showed that the increase of intraocular pressure (IOP) in glaucomatous eye is associated with the increasing resistance to aqueous humor outflow effects of transforming growth factor-β (TGF-β) and CD44.Qingguangan is a traditional Chinese medicine and used to treat glaucoma.However,its mechanism of lowing-IOP effect is not elucidated.Objective This study was to investigate the lowing-IOP effect and mechanism of qingguangan granule in DBA/2J mouse,a spontaneous glaucoma model mice.Methods Ten 3 month-old female DBA/2J mice with normal IOP were chosen as control group,and 20 spontaneous ocular hypertension mice aged 9 months were randomized into high IOP group and qingguangan-treated group,with 10 mice for each group.The qingguangan (2.5 g/kg) was administered by gavaging twice per day for consecutive 15 days in the qingguangan-treated group,and normal saline solution was used in the same way in the control group and high IOP group.IOP was measured by anterior chamber injection/suction system at a perfusion rate of 2.5 and 5.0 μl/min,respectively,and the coefficient of aqueous outflow facility (C value) and outflow resistance (R value) were calculated.Another 60 3-month-old DBA/2J mice were randomized into blank control group gavaged with normal saline solution and high-,middle-and low-dose qingguangan groups gavaged with 25.00,12.50 and 6.25 g/kg drugs,respectively,and the mouse serum containing drugs was extracted 7 days after treatment.The scleral tissue with trabecular meshwork were obtained for the culture of trabecular meshwork cells and the cells were identified by immunohistochemistry of fibronectin (FN),laminin (LN) and neuronspecific enolase (NSE).TGF-β was added into the medium for 24 hours with the final concentration of 0,5,10,20,50 and 100 ng/ml,and MMT chromatometry was employed to detect the cell vitality.The cells pre-treated with 20 ng/ml TGF-β were treated with different concentration of drug serum for 24,48 and 72 hours,and the level of TGF-β2 receptor in cell supernatant and the expression of CD44 protein in the cells were detected by ELISA and Western blot assay,respectively.Results The IOPs with perfusion both 2.5 μl/min and 5.0μl/min in the qingguangan-treated group and the control group were significantly lower than those in the high IOP group (all at P＜0.01).Compared with the high IOP group,the C value was significantly reduced (2.35±1.34 vs.1.08±0.36) and the R value was evidently elevated (0.64±0.55 vs.1.05± 0.47) in the qingguangan-treated group (all at P＜0.01).Cultured cells were spindle-shaped with the positive response to FN,LN and NSE antibody.The cell vitality was lower in the 5,10 and 20 ng/ml TGF-β group than that in the 0 ng/ml TGF-β group (all at P＜0.05).Compared with the blank control group,the TGF-β2 receptor content in the supernatant and the related expression level of CD44 protein in the cells were elevated in the TGF-β-treated group (all at P＜0.01),and TGF-β2 receptor contents and CD44 expression levels in the TGF-β+high dose drug serum group was significantly lower than those in the TGF-β group and TGF-β +low dose drug serum group 24,48 and 72 hours after culture (all at P＜0.01).Conclusions Qingguangan can lower IOP of spontaneous glaucoma mice by affecting aqueous humor dynamics.Serum containing qingguangan down-regulates the expressions of TGF-β2 receptors and CD44 in trabecular meshwork cells in vitro.

Ocular findings and histological change in glaucoma model of DBA/2J Mice / 眼科研究

Background As a hereditary chronic glaucoma model,DBA/2J mouse is widely used in the experimental research of glaucoma worldwide.Although some researches have determined the ocular change induced by hypertention of DBA/2J mouse,there is seldom reports about the research on the relationship of ocular pathological abnormality and development course in DBA/2J mouse.Objective The present study is to characterize the ocular abnormalities and histological changes induced by hypertention in different ages of DBA/2J mice.Methods The clean female DBA/2J mice aged 3-,5-,7-,9-,11-,14-month-old (6 mice for each) were used in this study,and age matched 18 female C57BL/6J mice were as controls.Intraocular pressure (IOP) of mice was measured by anterior chamber puncture of microneedle.The animals were sacrificed and retinal flat mounts were prepared for histopathological examination under the light microscope.Retinal ganglion cells (RGCs) were counted by retinal Nissl staining.Morphology of optic nerve cup in frozen section was examined under the light microscope.The experiment followed the Standard of Association for Research in Vision and Ophthalmology.Results Developing pigment dispersion,iris stroma atrophy,transillumination defects and pupil deformation were found in DBA/2J mice.IOP began to rise in 7-month-old DBA/2J mouse,peaked in 9-month-old mouse and returned to normal in 14-month-old DBA/2J mouse.A significant difference was found in IOP among different ages of DBA/2J mice (F=27.600,P＜0.05) but not C57BL/6J mice (F=0.249,P=0.781).RGCs loss was observed in 7-month-old DBA/2J mice and more serious from 9 to 11-month-old mice,showing a significant decline of RGCs among different ages of DBA/2J mice (F=23.594,P=0.000) but not C57BL/6J mice (F=1.816,P=0.211).The abnormality of optic nerve cupping and decrease of retinal nerve fiber layer were found in 9 to 14-month-old months old DBA/2J mice and were normal in age-matched C57BL/6J mice.Conclusion The abnormal alteration of the ocular anterior segment in DBA/2J mouse is gradually aggravated with aging.The findings of eye in DBA/2J mouse is characterized by secondary glaucoma.DBA/2J mouse offer an ideal animal model for the research of glaucoma.

Changes in Retinal Neuronal Population in DBA/2J Mice with Topical Intraocular Pressure Lowering Drugs

PURPOSE: The DBA/2J (D2) mouse is a transgenic mouse with pigmentary glaucoma. In a previous study, we found a reduction of inner retinal thickness in D2 mice. We attempted to discover the effect of eye drops on the retina of D2 mice. METHODS: Ten-month-old D2 mouse eyes were treated with Timoptic XE(R), Cosopt(R), and Xalacom(R) eye drops for a 1-month period. Immunohistochemical staining was performed on the mouse eye sections for analysis. RESULTS: In the control group, GABA and OPN immunoreactivity were markedly decreased and NOS immunoreactivity was increased. In all experimental group, GABA and OPN immunoreactivity were increased, and OPN immunoreactivity was markedly increased especially in the Cosopt(R) group. NOS immunoreactivity was decreased in all experimental groups. There was no difference in glycine immunoreactivity between the control and experimental groups. CONCLUSIONS: Combination anti-glaucoma eye-drops to the D2 mouse changed the retinal neuronal population and these drugs might play an important role in the mechanisms of retinal neuronal death; potential strategies for neuroprotection should therefore be evaluated.

Histologic Characteristics in the Eyes of DBA/2J Mice according to the Degree of Intraocular Pressure

PURPOSE: We assessed the correlation between ocular abnormalities and the degree of intraocular pressure (IOP) in the DBA/2J (D2) transgenic mice which were proven to have pigmentary dispersion syndrome and developing glaucoma. METHODS: Nine-months-old D2 mice were examined with biomicroscopy under anesthesia and measured for IOP by Tono-Pen, Hematoxylin and eosin staining was performed on the eye sections of the mice to analyze differences between the low-grade IOP group and the high-grade IOP group. RESULTS: Ocular abnormalities including iris pigment loss, iris transillumination, iris stromal atrophy, anterior synechia, thinning of the retina, and ganglion cell loss were found; all of which appeared to be pressure- dependent. CONCLUSIONS: These results corroborate that both IOP and age might be considered for studies using D2 mice, and suggest that D2 mice are a useful glaucoma model to study the mechanisms of retinal ganglion cell death and to evaluate strategies for neuroprotection.