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Objective:To compare the effects of different intraocular infusion solutions on histology and function of retina.Methods:Human corneal endothelial cells (HCEC), human retinal pigment epithelium (HRPE) cells and rat retinal ganglion cells (RGC) were divided into normal control group, balanced saline solution (BSS) group and compound electrolyte intraocular irrigating solution (CEIIS) group, and the cells were cultured in 10% DMEM/F12 medium, BSS and CEIIS for 12, 24 and 48 hours, respectively, according to grouping.The proliferation absorbance value of cultured cells was measured by cell counting kit-8 (CCK8) method.The expression of apoptosis related proteins in cultured cells was detected by cellular immunofluorescence staining.The cell apoptosis rate and cell cycle were measured by flow cytometry.The mitochondrial damage was detected by lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH) quantitative detection kit.Fifteen New Zealand white rabbits were randomly divided into control group ( n=3), BSS group ( n=6) and CEIIS group ( n=6). The left eyes were taken for vitrectomy and different intraocular perfusion fluids were used during vitrectomy according to grouping.The retinal function of operative eyes was measured by flash electroretinogram (ERG) before operation and 24 hours after operation, and the structural changes of each layer of retina were detected by optical coherence tomography (OCT). The early apoptosis of retinal cells was detected by TUNEL staining.The expressions of cytochrome C and bax protein in retina were detected by immunohistochemical staining.The ultrastructural changes of retina were observed under a transmission electron microscope.The use and care of animals complied with the ARVO statement.This study protocol was approved by an Ethics Committee of Peking University People's Hospital (No.2019PHE059). Results:The three kinds of cultured cells in BSS and CEIIS groups were damaged in various degrees.With the extension of culture time, proliferated cells were decreased and the number of apoptotic cells was increased.Compared with the BSS group, cultured cells in the CEIIS group were dense and in orderly arrangement with uniform morphology and size.The apoptosis rates of HRPE cells and RGC in the BSS group were (37.157±6.918)% and (29.993±12.330)%, respectively, which were significantly higher than (4.163±1.310)% and (6.337±1.903)% in the CEIIS group ( P=0.003, 0.045). There was no significant difference in G0/G1+ S phase ratio of HCEC and HRPE cells among the normal control group, BSS group and CEIIS group (HCEC: F=2.226, P=0.189; HRPE: F=2.634, P=0.151), and the proportion of G2/M division arrest phase of RGC in the BSS group was significantly higher than that in the normal control group and CEIIS group ( P=0.047, 0.024). The proliferation absorbance values of HCEC, HRPE cells and RGC in the CEIIS group were significantly higher than those in the BSS group at each culture time point (all at P<0.05). The fluorescence intensity of cytochrome C, bax, caspase-3 and caspase-9 proteins in the BSS group was stronger than that in the normal control group and CEIIS group, and the fluorescence intensity of bcl-2 was weaker than that in the CEIIS group, and the fluorescence intensity of zonula occluden-1 (ZO-1) was weaker than that in the normal control group and CEIIS group.The release level of LDH in the BSS group was significantly higher than that in the CEIIS group at different time points (all at P<0.001). After 48 hours of culture, the release level of SDH in the BSS group was significantly higher than that in the CEIIS group ( P<0.05). No retinal histological abnormalities was found through OCT examination of rabbit eyes after vitrectomy in the two groups, but transmission electron microscopy showed that there were different degrees of loose arrangement of retinal photoreceptor cells, a large number of photoreceptor outer membrane discs falling off and vacuolar degeneration in the two groups, especially in the BSS group.TUNEL staining showed that the apoptotic cells were mainly located in the inner nuclear layer and RGC layer.The number of apoptotic retinal cells was (135.2±22.8)/high-power field of vision in the BSS group, which was significantly higher than (81.3±17.7)/high-power field of vision in the CEIIS group ( t=4.175, P=0.002). Full field flash ERG showed that the amplitudes of scotopic 3.0 ERG a- and b-wave in the CEIIS group after operation were significantly lower than those before operation, but the differences were not statistically significant (all at P>0.05). The amplitudes of scotopic 3.0 ERG a- and b-wave in the BSS group after operation were significantly lower than those before operation ( P=0.026, 0.010). Conclusions:In vivo and in vitro research results show that compared with BSS, there were few apoptotic cells in retinal tissue after vitrectomy perfused by CEIIS.
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Retinal vein occlusion (RVO) is the second visual threatening retinal disorders followed by diabetic retinopathy in the elderly.In the past decades,increasing knowledge of the natural history,aetiology and risk factors,medical management investigation,together with the support of high level evidence-based medical evidence and the results of real-world clinical trials play key roles in guiding the clinical practice.However,without understanding the pathogenesis and pathogeny of the disease,it is difficult to implement a comprehensive,precise and personalized treatment strategy for the RVO patients.It is of significance in the clinic to discuss the pathological process of RVO,analyze the etiological characteristics of the disease,reveal the clinical outcomes,which aim to facility the optimal treatment and follow-up procedure for the patients.
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Background Choroidal neovascularization (CNV) is the primary pathogenic cause of many fundus diseases.Oxidative stress injury of retinal pigment epithelial (RPE) cells plays important role in angiogenesis of choroid new blood vessels.Oxidative stress injury can active p75NTR receptor, a member of tumor necrosis factors family,resulting in the proliferation of vascular endothelial cells.However, the mechanisms of vascular endothelial cell proliferation remain unclear.Objective This study was conducted to investigate the effect of p75NTR overexpression on CNV and the relative mechanism.Methods The ARPE-19 cell line was used in this study.RPE cells were transfected with p75NTR receptor overexpressed plasmid, and untransfected cells served as the control group.The transfected results were verified by reverse transcription-PCR and Western blot assay.Viability of the cells over time was determined in the p75NTR receptor plasmid transfected group by using BrdU assay.The percentage of apoptotic cells was detected by flow cytometry using Annexin V-FITC/PI fluorescence staining.The percentage of reactive oxygen species (ROS) expression in the cells was detected by using H2 DCFDA fluorescence and flow cytometry.Mitochondrial membrane potential and cytochrome C expression were examined under the confocal microscope.The protein expressions of cleaved caspase-3, Fas and VEGF were determined by Western blot assay.Results The relative expression level of p75 NTR receptor mRNA was (6.11 ±0.77) times higher than that of the control group, and relative expression level of p75NTR receptor protein in the cells in the p75NTR receptor plasmid transfected group was (7.42±0.48) times higher than that in the control group (t=11.49 and 23.17 ,both at P＜0.01).The absorbency values of the p75NTR receptor plasmid transfected group were (93.12±0.56) ％ , (86.30±0.66) ％ , (72.53-±0.86) ％ and (60.77 ±2.81) ％ in 12,24,36 and 48 hours after plasmid transfection, which were significantly lower than 100％ in the control group, and the apoptotic percentages were evidently higher than that in the control group (all at P＜0.05).The relative fluorescence intensity of ROS fluorescence in the p75NTR receptor plasmid transfected group was 2.4 times higher than that in the control group,showing significant difference (t=16.45, P＜0.01).The positive expressing rate of mitomarker (mitochondrial membrane potentials) was 100％ in the control group and (37.30± 2.06)％ in the p75NTR receptor plasmid transfected group, with significant difference between them (t =57.71,P＜0.01).The fluorescence intensity of cytochrome C expression was elevated in the p75NTR receptor plasmid transfected group compared with the control group.Compared with the control group,the expressing levels of cleaved caspase-3 ,Fas and VEGF165 proteins in the cells were significantly raised in the p75NTR receptor plasmid transfected group (all at P＜0.01).Conclusions Overexpression of p75NTR receptors in RPE cells leads to mitochondrial damage and cellular apoptosis and the secretion of VEGF protein, which sequentially promote CNV.P75NTR receptor may be another important regulation pathway in RPE oxygen damage.
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Choroidal neovascularization (CNV) is the key characteristic of neovascular age-related macular degeneration (nAMD),and the effective therapy is intravitreal injection of anti vascular endothelial growth factor (VEGF) agents based on clinical and basic research.In the meantime the challenge is how to further improve the inhibiting effect for CNV and visual function of anti-VEGF treatment on nAMD.The new strategy and drug delivery devices for anti-VEGF treatment will optimize the clinical scheme.From bench to bedside,the research on targeted treatment of angiogenesis brings the bloom of nAMD medical therapy.
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Background There remains some controversy over whether polypoidal choroidal vasculopathy (PCV) represents a subtype of neovascular age-related macular degeneration (nAMD).Anti-vascular endothelial growth factor (VEGF) therapies are important in the treatment of PCV and nAMD.It has been identified that VEGF-A was differentially spliced from exons 8 and formed two isoforms families:the pro-angiogenic VEGFxxx family and the anti-angiogenic VEGFxxxb family.However,the role of the two VEGF families in PCV and nAMD was still unclear.Objective This study was to measure the contents of pro-angiogenic VEGFxxx family and the anti-angiogenic VEGFxxxb family in aqueous humor of nAMD and PCV patients and explore their effect on nAMD and PCV.Methods Thirty-four nAMD patients and 26 PCV patients were enrolled in Peking University People's Hospital during March to December,2013,and 16 age-related cataract patients served as controls.The aqueous humor samples 0.1 ml was collected before the introvitreous injection of anti-VEGF drug.The contents of pro-angiogenic VEGFxxx family and the anti-angiogenic VEGFxxx b family in the aqueous humor were measured by enzyme-linked immunosorbent assay(ELISA).Results The concentrations of VEGF in the aqueous humor in the nAMD group,PCV group and control group were (4 210.00±998.40),(387.00±51.31) and (377.40 ±69.97)pg/ml,respectively,showing a significant difference among the three groups (F =12.851,P =0.000).The concentrations of VEGF165 b in the aqueous humor in the nAMD group,PCV group and control group were (205.50±12.59),(159.40±16.25) and (347.90±29.18) pg/ml,with a significant difference among them (F=23.752,P=0.000).Compared with the control group,VEGF content in the aqueous humor was elevated and the VEGF165b content was declined in the nAMD group,and VEGF165b was lowed in the PCV group,with significant differences between them(all at P=0.000).However,no significant difference was seen in the change of VEGF between the PCV group and the control group (P=0.992).The VEGF content in the aqueous humor was higher in the nAMD group than that in the PCV group (P =0.001),but VEGF165b content was insignificantly different (P =0.097).Conclusions The downregulation of VEGFxxx b may be associated with nAMD and PCV.The different role of VEGFxxx b in the development of PCV and nAMD needs to be verified in further studies.
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<p><b>BACKGROUND</b>Glaucoma, an irreversible optic nerve neuropathy, always results in blindness. This study aimed to evaluate glaucoma-like features in the rat episcleral vein cauterization (EVC) model by multiple in vivo and in vitro evidences.</p><p><b>METHODS</b>Wistar rat was used in this study. The elevated intraocular pressure (IOP) was induced by cauterization of three episcleral veins. IOP was monitored with Tono-Pen XL tonometer. Time-dependent changes to the neuronal retinal layers were quantified by Fourier domain-optical coherence tomography. The function of retina was evaluated by electroretinogram (ERG). Survival of retinal ganglion cells (RGCs) was quantified by retrograde labeling. Histology study was performed with retinal sections stained with hematoxylin-eosin, glial fibrillary acidic protein, and neuronal nuclear antigen. Retina and aqueous humor protein were extracted and cytotoxic protein tumor necrosis factor alpha (TNF-α) and alpha-2 macroglobulin (α2m) were measured with Western blotting.</p><p><b>RESULTS</b>EVC is a relatively facile intervention, with low failure rates (<5%). After surgical intervention, chronic mild IOP elevation (about 1.6-fold over normal, P < 0.05) was induced for at least 6 weeks without requiring a second intervention. High IOP causes chronic and progressive loss of RGCs (averaging about 4% per week), progressive thinning of neuronal retinal layers (3-5 μm per week), and reduction of a- and b-wave in ERG. EVC method can also induce glial cell activation and alterations of inflammation proteins, such as TNF-α and α2m.</p><p><b>CONCLUSION</b>EVC method can establish a robust, reliable, economic and highly reproducible glaucomatous animal model.</p>