Neural stem cell is a kind of stem cells that can differentiate into neural and glial cells. While Müller cells, the main endogenous neural stem cell in retina,have the features to reentry into the cell cycle and differentiate into neural cells after retinal damage. Although it is highly effective for retinal Müller cell differentiation spontaneously after retinal injury in vertebrates, this feature is rigorous restricted in mammals. Recently, some transcription factors,such as Ascl1, Sox2, Lin28, Atoh7, are sufficient to drive quiescent Müller cells back in proliferation to generate new retinal neurons. Moreover, combining Ascl1 expression with a histone deacetylase inhibitor can bypass the limitation and increase the generation of new neurons in the adult retina. These regenerated neurons integrate the existing neuronal network and are able to respond to light, indicating that they can likely be used to restore vision. While these results are extremely promising, the regenerative response is still limited, likely because the proliferative capacity of mammalian Müller cells is low compared to their zebrafish counterparts. It is indeed necessary to identify new factors increasing the efficiency of the regenerative response.
ObjectiveTo analyze the risk factors of neovascular glaucoma (NVG) after 25G pars plana vitrectomy (PPV) in proliferative diabetic retinopathy (PDR).MethodsA retrospective study. From January 2017 to December 2018, 340 PDR patients (340 eyes) with vitreous hemorrhage (VH) who were first treated with PPV in Tianjin Medical University Eye Hospital were included in the study. Among them, 185 were male and 155 were female, with an average age of 55.79±10.82 years. The duration of diabetes was 13.01±7.70 years, the fasting blood glucose was 7.55±2.15 mmol/L. Nineteen patients combined coronary heart disease, and 20 patients combined cerebral infarction. All patients underwent best-corrected visual acuity (BCVA), intraocular pressure (IOP), non-contact fundus examination, and fundus color photographs. BCVA was measured using an international standard Snellen visual acuity chart, and the values were converted to logarithm of the minimum angle of resolution (logMAR) scores for data analysis. The baseline logMAR BCVA was 2.04±0.73, The baseline IOP was 15.45±2.93 mmHg (1 mmHg=0.133 kPa). The duration of VH was 2.98±1.46 months, ranged from 3 weeks to 6 months. Three hundred and forty eyes included 93 eyes of PDR Ⅳ stage (27.35%), 107 eyes of Ⅴ stage (31.47%), and 116 eyes of Ⅵ stage (34.12%), combined tractional retinal detachment (TRD) 83 eyes. All patients underwent 25G standard three channel vitrectomy through the pars plana of the ciliary body. Preoperative anti-VEGF injection was performed in 57 eyes, internal limiting membrane (ILM) peeling in 234 eyes, combined phacoemulsification cataract surgery in 262 eyes and 141 eyes intravitreal anti-VEGF injection at the end of surgery. The patients were followed up for at least 12 months, with an average follow-up time of 10.80±5.79 months. NVG was defined as the presence of neovascularization in the anterior chamber angle or iris with an IOP higher than 21 mmHg after vitrectomy. Kaplan-Meier method and Cox univariate and multivariate regression were used to analyze the relationship between baseline factors, ocular factors, surgical factors and the occurrence of NVG after surgery.ResultsAmong 340 eyes, 66 eyes (19.41%) developed NVG after vitrectomy during 12 months of observation, NVG occurred from 6 to 335 days after surgery, and the mean period between vitrectomy and developing NVG was 98.00±5.79 days. The incidence of NVG was 11.50%, 15.29% and 20.75%, respectively in the 3rd, 6th and 12th month after PPV. The result of univariate analysis with the Cox regression analysis showed that the development of NVG at 12 months after surgery and age, combined coronary heart disease or cerebral infarction, combined with cataract phacoemulsification, ILM peeling, preoperative anti-VEGF injection had effect on postoperative NVG (P＜0.05). Ocular factors such as PDR staging, combined TRD, preoperative logMAR BCVA, preoperative intraocular pressure, etc. had no effect on the occurrence of NVG after surgery (P＞0.05). Combined cataract phacoemulsification surgery, internal limiting membrane peeling, surgical factors such as intracavity injection of anti-VEGF drugs 3 days before surgery, had an impact on the occurrence of NVG after surgery (P＜0.05). The meaningful variables of the Cox univariate analysis were incorporated into the multivariate Cox proportional hazard model for analysis, and the influencing factors of NVG after surgery were gradually regressed. The results showed that age, coronary heart disease or cerebral infarction, combined with phacoemulsification of cataract, and internal limiting membrane removal during surgery were independent risk predictors of NVG after surgery (P＜0.05).ConclusionsYounger, coronary heart disease or cerebral infarction, combined with cataract phacoemulsification are the risk factors of NVG in PDR patients after PPV. The removal of internal limiting membrane can reduce the incidence of NVG.
ObjectiveTo observe the safety and effectiveness of patching retinal breaks with Healaflow in 27G vitrectomy combined with air tamponade in the treatment of rhegmatogenous retinal detachment (RRD).MethodsClinical-based prospective continuous study. From March 2017 to May 2018, 51 eyes of 50 RRD patients diagnosed in Tianjin Medical University Eye Hospital were included in the study. All eyes were treated with 27G vitrectomy, and laser photocoagulation was performed around retinal hiatus and denaturation zone after complete retinal reattachment. A blunt 27G needle was used to completely cover the surface of the retinal tear with the Healaflow. The injection amount was determined according to the size of the retinal tear, and the standard was that the tear was completely contained. There was no postoperative position limitation. The average follow-up was 15.8±6.3 months. The primary and final anatomic attachment rate, BCVA after operation, the intraoperative and postoperative complications, the recurrence of retinal detachment and so on were recorded.Results51 eyes of 50 patients were enrolled, including 29 males (58.0%) and 21 females (42.0%). The average age was 58.5±1 years. A single break was present in 28 eyes (54.9%) and 2 to 5 breaks in 23 eyes (45.1%). The macula was involved in 32 eyes (62.7%) and attached in 19 eyes (37.3%) intraoperatively. Initial reattachment was achieved in 50 eyes (98.0%) and final reattachment in 51 eyes (100.0%). The logMAR BCVA before and 3 months after operation were 0.95±0.80 and 0.22±0.17, respectively. The difference of logMAR BCVA between before and after operation was significant (t=7.336, P＜0.001). The intraocular pressure was elevated transiently in 31 eyes (60.8%). No other complications occurred during follow-up.ConclusionThe treatment of primary RRD with 27G vitrectomy combined with Healaflow patch and air tamponade is a safe, effective and convenient method with high success rate and rapid recovery of visual function.
ObjectiveTo characterize proteomic profile in aqueous humor of patients with pathologic myopia (PM) using quantitative proteomic analysis, which may provide new clues to understand the mechanisms and possible treatments of PM.MethodsA cross-sectional study. From January 2019 to August 2019, aqueous humor samples (32 cataract patients) were collected for quantitative proteomic analysis using liquid chromatography tandem mass spectrometry at Tianjin Medical University Eye Hospital. There were 11 males and 21 females. They were 58-76 years old with an average age of 68.41±6.09 years old. Sixteen patients with PM were regarded as PM group, 16 patients without myopia were regarded as the control group. The aqueous humor samples (100-150 μl ) were collected from all patients before cataract surgery. Using protein quantification and non-labeled liquid chromatography tandem mass spectrometry analysis, differentially expressed proteins were obtained. Five different proteins were randomly selected for ELISA verification. The differentially expressed proteins were further analyzed by gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes, which were validated using ELISA in the other twenty samples of each group.ResultsA total of 583 proteins were identified and 101 proteins were found to be differentially expressed, including 63 up-regulated proteins and 38 down-regulated proteins. ELISA verification results showed that the expression trend of the 5 differentially expressed proteins between the PM group and the control group was consistent with the results of Label-free quantitative proteomics analysis. The main classifications of these differentially expressed proteins were protein-binding activity modulator, defense/immunity protein, protein modifying enzyme, metabolite interconversion enzyme, extracellular matrix protein, transfer/carrier protein and so on. The bioinformatics analysis suggested that PM was closely associated with inflammation and immune interactions, and remodeling of extracellular matrix.ConclusionsCompared with the control group, the protein expression profile of PM patients' aqueous humor specimens has obvious changes. These differences indicate that PM is closely related to inflammation and immune interaction and extracellular matrix remodeling.
ObjectiveTo compare and analyze the application of anti-vascular endothelial growth factor (VEGF) drugs for intravitreal injection in the real world before and after the establishment of one-stop intravitreal injection center, as well as the advantages and disadvantages of different management modes. MethodsA retrospective clinical study. A total of 4 015 patients (4 659 eyes) who received anti-VEGF drugs for ocular fundus diseases at the Tianjin Medical University Eye Hospital from July, 2018 to June, 2022 were included in the study. There were 2 146 males and 1 869 females. The ocular fundus diseases in this study were as follows: 1 090 eyes of 968 patients with wet age-related macular degeneration (wAMD); 855 eyes of 654 patients with diabetic macular edema (DME); 1 158 eyes of 980 patients with diabetic retinopathy (DR); 930 eyes of 916 patients with macular edema secondary to retinal vein occlusion (RVO-ME). A total of 294 eyes of 275 patients with choroidal neovascularization secondary to pathological myopia (PM-CNV); 332 eyes of 222 patients with other fundus diseases. A total of 13 796 anti-VEGF needles were injected. A total of 1 252 patients (1 403 eyes) from July 2018 to June 2020 were regarded as the control group. From July 2020 to June 2022, 2 763 patients (3 256 eyes) who received anti-VEGF treatment in the intravitreal injection center were regarded as the observation group. The total number of intravitreal injection needles, the distribution of anti-VEGF therapy in each disease according to disease classification, the proportion of patients who chose the 3+ on-demand treatment (PRN) regimen and the distribution of clinical application of different anti-VEGF drugs were compared between the control group and the observation group. The waiting time and medical experience of patients were investigated by questionnaire. χ2 test was used to compare the count data between the two groups, and t test was used to compare the measurement data. ResultsAmong the 13 796 anti-VEGF injections in 4 659 eyes, the total number of anti-VEGF drugs used in the control and observation groups were 4 762 and 9 034, respectively, with an average of (3.39±3.78) and (2.78±2.27) injections per eye (t=6.900, P＜0.001), respectively. In the control and observation groups, a total of 1 728 and 2 705 injections of anti-VEGF drugs were used for wAMD with an average of (5.14±4.56) and (3.59±2.45) injections per eye, respectively; a total of 982 and 2 038 injections of anti-VEGF drugs were used for DME with an average of (4.36±4.91) and (3.24±2.77) needles per eye, respectively. Additionally, a total of 942 and 2 179 injections of anti-VEGF drugs were injected for RVO-ME with an average of (3.98±3.71) and (3.14±2.15) injections per eye, respectively; a total of 291 and 615 injections of anti-VEGF drugs were injected for PM-CNV with an average of (3.31±2.63) and (2.99±1.69) injections per eye, respectively. A total of 683 and 1 029 injections of anti-VEGF drugs were injected for DR with an average of (1.60±1.26) and (1.41±1.05) injections per eye, respectively. The clinical application and implementation of "3+PRN" treatment were as follows: 223 (66.4%, 223/336) and 431 eyes (57.2%, 431/754) in the wAMD (χ2=8.210, P=0.004), 75 (33.3%, 75/225) and 236 (37.5%, 236/630) eyes in the DME (χ2=1.220, P＞0.05), and 97 (40.9%, 97/237) and 355 eyes (51.2%, 355/693) in the RVO-ME (χ2=7.498, P=0.006), 39 (44.3%, 39/88) and 111 eyes (53.9%, 111/206) in the PM-CNV ( χ2=2.258, P＞0.05), respectively. In addition, the results of the questionnaire survey showed that there were significant differences between the control and observation groups regarding the time of appointment waiting for surgery (t=1.340), time from admission to entering the operating room on the day of injection (t=2.780), time from completing preoperative treatment preparation to waiting for entering the operating room (t=8.390), and time from admission to discharge (t=6.060) (P＜0.05). ConclusionsThe establishment of a one-stop intravitreal injection mode greatly improved work efficiency and increased the number of injections. At the same time, the compliance, waiting time, and overall medical experience of patients significantly improved under centralized management.
ObjectiveTo observe the differences in the positive rate of conjunctival sac microbial culture after different methods of preventing infection before intravitreal injection (IVI). MethodsA prospective case-control study. A total of 1 200 participants with fundus diseases who received IVI injection at Tianjin Medical University Eye Hospital from July 2021 to December 2023 were included. Patients were randomly divided into 6 groups according to eye spot with antibiotic solution 3, 1 and 0 days before IVI and local eye disinfection with povidone-iodine (PVI) 3 min and 30 s before IVI: the first 3 days of antibiotics+3 min PVI group, the first 1 day of antibiotics+3 min PVI group, the first 0 days of antibiotics+3 min PVI group, the first 3 days of antibiotics+30 s PVI group, the first 1 day of antibiotics+30 s PVI group, the first 0 days of antibiotics+30 s PVI group, there were 200 cases in each group. Microbial sampling and cultivation of conjunctival sac were conducted before IVI to compare the differences in positive rates among different groups. Multiple group comparisons were conducted using one-way analysis of variance; The comparison of count data is conducted using χ2 test. ResultsAmong the 1 200 patients, there were 566 males and 634 females. Age (62.59±13.44) years old. There were 397 cases of diabetes and 482 cases of hypertension. IVI frequency (2.35±2.34). 64 cases were positive for conjunctival sac culture before IVI. The age (F=1.468), sex composition ratio (χ2=2.876), diabetes (χ2=10.002), hypertension (χ2=6.019), times of IVI (χ2=4.507), and positive rate of conjunctival sac bacterial culture (χ2=6.272) of patients in each group had no statistical significance (P＞0.05). Using the duration of antibiotic application before IVI as a stratified factor, there was no statistically significant difference in the positive rate of conjunctival sac culture between groups with different durations of antibiotic application before IVI [χ2=0.414, P=0.52, combined odds ratio (OR)=0.819, 95% confidence interval (CI) 0.493-1.360]. Using the duration of PVI application as a stratified factor, there was no statistically significant difference in the positive rate of conjunctival sac culture between different PVI disinfection times [χ2=0.000, P=1.00, combined OR=1.00, 95% CI 0.503-1.988]. ConclusionsPre IVI treatment with 0.5% PVI for 30 s can inhibit the growth of microbial colonies in the conjunctival sac. The application of local antibiotic eye fluid in the anterior eye of IVI cannot reduce the positive rate of conjunctival sac bacteria.
ObjectiveTo observe the protective effect of polypyrimidine bundle-binding protein-related splicing factor (PSF) over-expression on RPE cell injury induced by advanced glycation end products (AGEs).MethodsThe human RPE cells cultured in vitro were divided into three groups: normal control group (N group), blank control group (N + AGEs group), empty vector control group (Vec + AGEs group), and PSF high expression group (PSF + AGEs). group). RPE cells in N group were routinely cultured; RPE cells in N + AGEs group were only transfected but did not introduce any exogenous genes combined with AGEs induction; Vec +AGEs group and PSF + AGEs group were transfected with pcDNA The empty vector or pcDNA-PSF eukaryotic expression plasmid was introduced into RPE cells and induced by AGEs. Except the N group, the other 3 groups of cells were transfected accordingly, and were stimulated with 150 μg/ml AGEs for 72 h after 24 h. HE staining and Hoechst 33258 staining were used to observe the effect of high PSF expression on the morphological changes of RPE cells; ROS level detection was used to analyze the effect of PSF high expression on the ROS expression of RPE cells induced by AGEs; MTT colorimetric method was used to detect the high PSF expression Effects on the viability of RPE cells; Western blot was used to detect the effects of different time and dose of PSF on the expression of heme oxygenase 1 (HO-1).ResultsHE staining and Hoechst 33258 staining observation showed that the cells in group N were full in shape, the nucleus was round, the cytoplasm was rich, and the staining was uniform; the cells in N + AGEs group and Vec + AGEs group were reduced in size, the eosinophilic staining was enhanced, and the nucleus was densely densely stained. Pyrolysis and even fragmentation; the morphology of cells in the PSF + AGEs group was still full, the cytoplasm staining was more uniform, and the nucleus staining was uniform. The results of MTT colorimetry showed that high expression of PSF can effectively improve the viability of RPE cells, but this effect can be effectively antagonized by ZnPP, and the difference is statistically significant (F=33.26, P＜0.05). DCFH-DA test results showed that compared with the N + AGEs group and Vec + AGEs group, the ROS production in PSF + AGEs group decreased, the difference was statistically significant (F=11.94, P＜0.05). Western blot analysis showed that PSF protein up-regulated HO-1 expression in a time- and dose-dependent manner. The relative expression level of HO-1 at 24, 48, and 72 h after PSF protein was significantly higher than that at 0 h, and the difference was statistically significant (F=164.91, P＜0.05). The relative expression level of HO-1 under the action of 0.1, 0.5, 1.0, 1.5, and 2.0 μg PSF protein was significantly higher than 0.0 μg, and the difference was statistically significant (F=104.82, P＜0.05).ConclusionPSF may inhibit the production of ROS by up-regulating the expression of HO-1, thus protecting the RPE cells induced by AGEs.
ObjectiveTo investigate the inhibitory effect of lentivirus-mediated polypyrimidine bundle binding protein-associated splicing factor (PSF) on retinal neovascularization (RNV) in mice model of oxygen-induced retinopathy (OIR).MethodsOne hundred and twelve 5-day-old C57BL/6J mice were randomly divided into normal control group, simple OIR model group, OIR model + lentivirus empty vector treatment group (Vec group) and OIR model + PSF lentivirus treatment group (PSF group), with 16, 32, 32 and 32 mice, respectively. When the mice were 7 days old, the mice in the normal control group were fed in a routine environment, and the mice in the OIR model group, Vec group and PSF group were established OIR model. The mice in the Vec group and PSF group were given an intravitreal injection of 1 μl of lentiviral vector and PSF lentivirus (titer 1×1011 TU/ml) at the age of 12 days. No injection was performed in the normal control group and simple OIR group. RNV was evaluated by counting the number of pre-retinal neovascular cells and analysis of non-perfusion area by immunofluorescent staining of the mouse retina. Real-time quantitative PCR was applied to detect the mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1). Western blot analysis was applied to detect the protein expression of Nrf2, HO-1 and PSF. Results Of the normal control group, simple OIR model group, Vec group and PSF group, the number of pre-retinal neovascular cell nuclei were 0.00, 14.36±5.50, 15.67±4.96, 8.13±2.09, the non-perfusion area were 0.00%, (35.71±2.81)%, (36.57±4.53)%, (15.33±4.75)%, respectively. The differences of the number of pre-retinal neovascular cell nuclei and non-perfusion area among 4 groups were significant (F=24.87, 165.70; P＜0.05). Compared with the normal control group, there were more pre-retinal neovascular cell nucleis and larger non-perfusion area in the simple OIR model group and Vec group (P＜0.05). Compared with the simple OIR model group and Vec group, there were lower pre-retinal neovascular cell nucleis and smaller non-perfusion area in the PSF group (P＜0.05). Real-time quantitative PCR and Western blot showed that the mRNA expression of Nrf2, HO-1 (F=53.66, 83.54) and protein expression of Nrf2, HO-1 and PSF (F=58.38, 52.69, 24.79) among 4 groups were significant (P＜0.05). The mRNA expression of Nrf2, HO-1 and protein expression of Nrf2, HO-1 and PSF in the simple OIR model group and Vec group decreased significantly than those in the normal control group (P＜0.05). The mRNA expression of Nrf2, HO-1 and protein expression of Nrf2, HO-1 and PSF in the PSF group were increased significantly than those in the simple OIR model group and Vec group (P＜0.05). model group and Vec group (P＜0.05).ConclusionIntravitreal injection of lentivirus-mediated PSF inhibits RNV in mice model of OIR possibly through up-regulating the expression of Nrf2 and HO-1.
ObjectiveTo observe the effect of pyrimidine bundle-binding protein-associated splicing factors (PSF) on the function of hypoxia-induced human retinal microvascular endothelial cells (hRMECs).MethodsA three-plasmid system was used to construct lentivirus (LV)-PSF. After LV-PSF infected hRMECs in vitro, the infection efficiency was measured by flow cytometry. Real-time quantitative PCR (RT-PCR) was used to detect the expression of PSF mRNA in hRMECs infected with LV-PSF. The experiment was divided into two parts, in vivo and in vitro. In vivo experiments: 20 healthy C57B/L6 mice at the age of postnatal 7 were randomly divided into normal group, oxygen-induced retinopathy (OIR) group, OIR+LV-Vec group, and OIR+LV-PSF group, each group has five mice. Mice in 3 groups were constructed with OIR models except the normal group and the mice in OIR group were not treated. The mice in the OIR + LV-Vec group and the OIR+LV-PSF group were injected with an empty vector (LV-Vec) or LV-PSF in the vitreous cavity, respectively. The effect of LV-PSF on the formation of retinal neovascularization (RNV) was observed then. In vitro experiments: hRMECs were divided into normal group, hypoxia group, vector group, and PSF high expression group. HRMECs in the normal group were cultured in vitro; hRMECs in the hypoxic group were restored to normal culture conditions for 3 h after 3 h of hypoxia stimulation; hRMECs in the vector group and PSF high expression group were infected with LV-Vec and LV-PSF for 48 h, and hRMECs were returned to normal culture conditions for 24 h with hypoxia stimulation for 3 h. The effect of PSF on cell proliferation was observed by MTT colorimetry. Cell scratch test and Transwell migration experiment were used to observe the effect of PSF on cell migration ability under hypoxia stimulation. RT-PCR was used to observe the mRNA expression of HIF-1α, VEGF and PSF in each group of cells.ResultsThe LV-PSF of stably expressing PSF was successfully constructed. The infection efficiency was 97% determined by flow cytometry. The level of PSF mRNA in hRMECs infected with LV-PSF was significantly increased and detected by RT-PCR. In vivo experiments: The RNV area of the mice in the OIR group and the OIR + LV-Vec group was significantly increased compared to the normal group (t=18.31, 43.71), and the RNV area of the mice in the OIR + LV-PSF group was smaller than that in the OIR group (t=11.30) and OIR + The LV-Vec group (t=15.47), and the differences were statistically significant (P＜0.05). In vitro experiments: MTT colorimetry results showed that the proliferative capacity of hRMECs in the hypoxic group was significantly enhanced compared with the normal group (t=2.57), and the proliferative capacity of hRMECs in the PSF high expression group was significantly lower than that of the normal, hypoxic, and vector groups (t=5.26, 5.46, 3.73), the differences were statistically significant (P＜0.05). The results of cell scratch test showed that the hRMECs could be stimulated by the hypoxia stimulation for 3 hours to restore the normal condition for 24 hours or 48 hours (t=8.35, 13.84; P＜0.05). Compared with the vector group, cell migration rate in the PSF-high expression group was not significant (t=10.99, 18.27, 9.75, 8.93, 26.94, 7.01; P＜0.05). Transwell experiments showed that the number of cells stained on the microporous membrane was higher in the normal group and the vector groups, while the number of cells stained in the PSF high expression group was significantly reduced (t=9.33, 6.15; P＜0.05). The results of RT-PCR showed that the mRNA expression of HIF-1α and VEGF in hRMECs in the hypoxic and vector groups increased significantly compared with the normal group (t=15.23, 21.09; P＜0.05), but no change in the mRNA expression of PSF (t=0.12, 2.15; P>0.05); compared with the hypoxia group and the vector group, the HIF-1α and VEGF mRNA expression in hRMECs in the PSF high expression group were significantly decreased (t=10.18, 13.10; P＜0.05), but the PSF mRNA expression increased (t=65.00, 85.79; P<0.05).ConclusionPSF can reduce the RNV area in OIR model mice. PSF may inhibit hypoxia-induced proliferation and migration of hRMECs through the HIF-1α/VEGF signaling pathway.