Objective To develop a method for the primary culture of retinal Muuml;ller cells of adult rabbit in vitro. Methods Retina was isolated from adult rabbit, cut into 1 mm times; 1 mm pieces, and placed into Dulbecco modified Eagle medium/F12 containing 20% fetal bovine serum to culture. Cultured cells were identified by inverted phase contrast microscope, transmissim electron microscope and immunohistochemistry staining method. Results Visible cell processes grew out from the retinal tissues after three days culture, and more cells grew radically around the retina after seven days culture. The cultured cells were often inflated at one side and had one long process at another side, and the nuclei were elliptical and there were two or more than two nucleoli under inverted phase contrast microscope. The cytoplasm was rich and contained abundant microfilaments in eight to ten nanometers under transmission electron microscope. Immunohistochemistry assay showed that 95% of the cells were positive for glial fibrillary acidic protein and cellular retinaldehydebinding protein. Conclusion Rabbit retinal Muuml;ller cells can be cultured by the explant culture method.
Objective To investigate the effect of astragaloside A (AS-A) on the photoreceptor degeneration induced by sodium iodate (NaIO3) and its related mechanism. MethodsSixty healthy male C57BL/6J mice, aged 6-8 weeks, were randomly divided into normal control (NC) group, NaIO3 group, and AS-A group, with twenty mice in each group. 30 min before modeling, AS-A group mice were intraperitoneally injected with 100 μl AS-A at a dose of 100 mg/kg body weight. 30 min later, mice in NaIO3 group and AS-A group were intraperitoneally injected with 100 μl NaIO3 at a dose of 30 mg/kg body weight. Subsequently, AS-A group mice were administered AS-A twice daily at 12 h intervals until the end of the experiment. On day 1 post-modeling, zonula occludens-1 (ZO-1) immunohistochemistry was performed to observe the structure of retinal pigment epithelium (RPE) cells; real-time quantitative polymerase chain reaction (qPCR) was conducted to detect the mRNA expression of various retinal chemokine ligand-2 (Ccl2), interleukin-1 beta (Il-1β), mixed lineage kinase domain-like protein (Mlkl), receptor-interacting protein kinase 3 (Ripk3), and tumor necrosis factor (Tnf). On day 3 post-modeling, immunohistochemistry was performed to observe the expression of ionized calcium binding adaptor molecule 1 (Iba1) and glial fibrillary acid protein (GFAP) in the retina; TdT-mediated dUTP nick-end labeling (TUNEL) assay was used to detect photoreceptor cell death in each group. On day 4 post-modeling, fundus morphology of mice in each group was observed by fundus color photography and optical coherence tomography (OCT). Hematoxylin-eosin staining (HE) was used to observe the morphological structure of the retina in each group. Inter-group comparisons between two groups were conducted using independent samples t-test, while comparisons among three groups were performed using one-way ANOVA. ResultsFundus color photography and OCT examination showed that a large number of scattered yellow-white subretinal nodular structures in the fundus of NaIO3 group mice, and a large number of strong reflection areas in the RPE layer. The number of strong reflection areas in the RPE layer was reduced in the AS-A group. Immunohistochemical analysis of ZO-1 showed that ZO-1 was largely lost on the RPE cell membrane in that NaIO3 group; whereas in the AS-A group, ZO-1 was evenly distributed on the RPE cell membrane. HE staining results showed circular black deposits were visible in the RPE layer of the NaIO3 group, and the inner and outer segments of photoreceptors were severely damaged, with a significant decrease in the number of outer nuclear layer (ONL) cell nuclei; whereas in the AS-A group, the RPE layer pigments were orderly, the inner and outer segments of photoreceptors were intact, and the number of ONL cell nuclei significantly increased. The results of TUNEL staining show that numerous TUNEL-positive cell nuclei were observed in the ONL of the retina in the NaIO3 group, while the number of TUNEL-positive cell nuclei in the ONL of the retina was significantly reduced in the AS-A group, with statistically significant differences (t=2.66, P＜0.05). The analysis of qPCR data showed that compared with the AS-A group, the relative expression levels of Mlkl, Ripk3, Ccl2, Il-1β and Tnf mRNA in the retina were significantly increased in the NaIO3 group, with statistically significant differences (F=39.18, 10.66, 53.51, 41.40, 24.13; P＜0.001). Immunohistochemical staining results showed that compared with NC group and AS-A group, the positive expression of GFAP in retina of NaIO3 group was significantly increased, and the difference was statistically significant (F=9.62, P＜0.05). ConclusionAS-A antagonizes NaIO3-induced photoreceptor degeneration in part by inhibiting photoreceptor cell death and neuroinflammation. Meanwhile, AS-A treatment protects against NaIO3-triggered perturbation of retinal homeostasis.
ObjectiveTo observe the expression of probucol on high glucose-induced specificity protein 1(SP1), kelchlike ECH associated protein1 (Keap1), NF-E2-related factor 2 (Nrf2) and glutamate-cysteine ligase catalytic (GCLC) in the cultured human müller cells and preliminary study the antioxidation of the probucol on müller cells.MethodsPrimary cultured human müller cells were randomly divided into four groups: normoglycaemia group (5.5 mmol/L glucose), normoglycaemia with probucol group (5.5 mmol/L glucose+100 μmol/L probucol), hyperglycemia group (25.0 mmol/L glucose), hyperglycemia with probucol group (25.0 mmol/L glucose + 100 μmol/L probucol). Immunofluorescence staining was used to assess distribution of SP1, Keap1, Nrf2, GCLC in human Müller cells. SP1, Keap1, Nrf2 and GCLC messenger RNA (mRNA) expression was evaluated by quantitative real-time RT-PCR (qRT-PCR). Independent sample t test was used to compare the data between the two groups.ResultsAll müller cells expressed glutamine synthetase (＞95%), which confirmed the cultured cells in vitro were the purification of generations of müller cells. The expressions of SP1, Keap1, Nrf2, and GCLC protein were positive in human müller cells. qRT-PCR indicated that SP1 (t=28.30, P＜0.000), Keap1 (t=5.369, P=0.006), and Nrf2 (t=10.59, P=0.001) mRNA in the hyperglycemia group increased obviously compared with the normoglycaemia group; GCLC (t=4.633, P=0.010) mRNA in the hyperglycemia group decreased significantly compared with the normoglycaemia group. However, SP1 (t=12.60, P=0.000) and Keap1 (t=4.076, P=0.015) in the hyperglycemia with probucol group decreased significantly compared with the hyperglycemia group; Nrf2 (t=12.90, P=0.000) and GCLC (t=15.96, P＜0.000) mRNA in the hyperglycemia with probucol group increased obviously compared with with the hyperglycemia group.ConclusionProbucol plays an antioxidant role by inhibiting the expression of SP1, Keap1 and up-regulating the expression of Nrf2, GCLC in müller cells induced by high glucose.
Objective To compare the effectiveness of talonavicular-cuneiform joint fusion with iliac bone grafting and without bone grafting in the treatment of Müller-Weiss diseases (MWD). Methods The clinical data of 44 patients (44 feet) with MWD who received talonavicular-cuneiform joint fusion between January 2017 and November 2022 and met the selection criteria was retrospectively analyzed. Among them, 25 patients were treated with structural iliac bone grafting (bone grafting group) and 19 patients without bone grafting (non-bone grafting group). There was no significant difference (P>0.05) in age, gender composition, body mass index, disease duration, affected side, Maceira stage, and preoperative American Orthopaedic Foot and Ankle Society (AOFAS) score, visual analogue scale (VAS) score, anteroposterior/lateral Meary angle, and Pitch angle between the two groups. Operation time, operation cost, and postoperative complications were recorded in the two groups. AOFAS and VAS scores were used to evaluate the function and pain degree of the affected foot. Meary angle and Pitch angle were measured on the X-ray film, and the joint fusion was observed after operation. The difference (change value) of the above indexes before and after operation was calculated for comparison between groups to evaluate the difference in effectiveness. Results The operation was successfully completed in both groups, and the incisions in the two groups healed by first intention. The operation time and cost in the bone grafting group were significantly more than those in the non-bone grafting group (P<0.05). All patients were followed up. The median follow-up time was 41.0 months (range, 16-77 months) in the non-bone grafting group and 40.0 months (range, 16-80 months) in the bone grafting group. There was skin numbness of the medial dorsalis of the foot in 1 case, internal fixation stimulation in 2 cases, and pain at the iliac bone harvesting area in 1 case of the bone grafting group. There was skin numbness of the medial dorsalis of the foot in 1 case and muscle atrophy of the lower limb in 1 case of the non-bone grafting group. There was no significant difference in the incidence of complications between the two groups (P>0.05). At last follow-up, the AOFAS scores of the two groups significantly improved when compared with those before operation, while the VAS scores significantly decreased, the anteroposterior/lateral Meary angle and Pitch angle significantly improved, and the differences were significant (P<0.05). There was no significant difference in the change values of outcome indicators between the two groups (P>0.05). There was no delayed bone union or bone nonunion in both groups, and joint fusion was achieved at last follow-up. Conclusion In the treatment of MWD, there is no significant difference in effectiveness and imaging improvement of talonavicular-cuneiform joint fusion combined with or without bone grafting. However, non-bone grafting can shorten the operation time, reduce the cost, and may avoid the complications of bone donor site.
ObjectiveTo investigate the short-term effectiveness of talonavicular joint arthrodesis and calcaneus osteotomy in the treatment of Müller-Weiss disease. MethodsBetween June 2015 and February 2017, 14 patients diagnosed Müller-Weiss disease, who were ineffective on conservative treatment, were treated with talonavicular joint arthrodesis and calcaneus osteotomy. There are 3 males and 11 females, with an average age of 46.2 years (range, 35-56 years). According to the Maceira grading criteria, 5 patients were rated as stage Ⅲ and 9 patients as stage Ⅳ. The disease duration ranged from 4 to 12 years (mean, 7 years). Preoperative X-ray films showed that all patients were not accompanied with adjacent joint arthritis. The hindfoot axis on Saltzman view was (9.8±2.8)°, calcaneal pitch angle (CPA) on lateral position was (14.7±5.1)°, Meary angle on lateral position was (4.8±2.8)°, and talar 1 meta-tarsal angle (T1MA) on anteroposterior position was (25.0±7.3)°. Preoperative visual analogue scale (VAS) score was 5.9±1.5, American Orthopedic Foot Ankle Society (AOFAS) ankle-hindfoot score was 58.8±17.6. ResultsAll patients were followed up 14-27 months (mean, 22.3 months). Medial numbness and incision infection occurred in 2, 2 cases, respectively. The other patients had no obvious discomfort. At last follow-up, VAS score was 1.6±1.3 and AOFAS score was 90.6±2.7, showing significant differences when compared with preoperative ones (t=8.18, P=0.00; t=–6.95, P=0.00). X-ray films showed that the talonavicular joint and calcaneus osteotomy achieved bony healing. The hindfoot axis on Saltzman view was (–2.5±2.7)°, CPA on lateral position was (25.0±5.2) °, Meary angle on lateral position was (2.6±2.1)°, T1MA on anteroposterior position was (8.1±3.8)°. There was no significant difference in Meary Angle between pre- and post-operation (t=1.53, P=0.15). And there were significant differences in the hindfoot axis, CPA, and T1MA between pre- and post-operation (t=11.93, P=0.00; t=–8.89, P=0.00; t=8.05, P=0.00). ConclusionFor Müller-Weiss disease patients without adjacent joint arthritis, who are ineffective on conservative treatment, the satisfied short-term effectiveness can be obtained when treated by talonavicular joint arthrodesis and calcaneus osteotomy.
ObjectiveTo observe the effect of polypyramidine tract binding protein-associated splicing factor (PSF) towards advanced glycation end products (AGEs) induced the apoptosis of Müller cells in vitro.MethodsExperimental study. Müller cells were cultured and divided into groups according to the project design, plasmid enhanced green fluorescent protein-PSF were transfected into the cells to achieve the overexpression of PSF Müller cells in vitro, then cells were exposed to AGEs and the Morphological changes were observed by HE staining and Hoechst 33258 staining while the survival rate of cells were detected by MTT assay. The effects of PSF on AGEs-induced Müller apoptosis was measured by Cell Death Detection ELISA kit. Meanwhile, 2′,7′-dichlorofluorescin diacetate staining was performed to monitor the protective effects of PSF on AGEs-induced Müller cells ROS.ResultsThe morphology of cells in normal group was full and the cytoplasm staining was uniform. In N+AGEs group and Vec+AGEs group, cell volume decreased, cytoplasm was dense and concentrated, and eosinophilic staining was enhanced. The cell morphology of PSF+AGEs group was still full, with uniform cytoplasm staining and uniform nucleus staining. The viability of N+AGEs group, Vec+AGEs group and PSF+AGEs group were 0.42±0.11, 0.35±0.12 and 0.68±0.12. The apoptosis values were 1.08±0.16, 0.96±0.20 and 0.44±0.08. The intracellular ROS levels were 28 833.67±3 550.06, 28 356.67±4 854.81, 186 163.00±382.54. Compared with N+AGEs group and Vec+AGEs group, the cell viability of PSF+AGEs group was significantly improved (F=20.65, P=0.000), cell apoptosis value (F=43.43, P=0.000) and intracellular ROS level (F=18.86, P=0.000).ConclusionPSF overexpression play a protective role in AGEs-induced apoptosis by inhibiting the production of ROS in Müller cells.
Objective To investigate the effect of methylprednisolone on the expression of glial fibrillary acidic protein (GFAP) and proliferating cell nuclear antigen (PCNA) in Müller cells of rats’ retinae injured by laser. Methods Forty SD rats were randomly divided into two groups and inflicted with laser photocoagulation.The rats in treatment group were given methylprednisolone by intraperitoneal injection with a dose of 30 mg/kg for 3 days.At the 3rd,7th,14th,and 28th day after photocoagulation respectively, the eyes were enucleated,fixed and cut into sections.Immunohistochemical examination was used to detect the expression of PCNA and GFAP. Results After photocoagulation the Müller cells expressed PCNA both in the treatment and control group,and the expression of PCNA decreased sharply after 3 days. The expression of PCNA in treatment group was less than that in control group. After photocoagulation the Müller cells also expressed GFAP and the expression of GFAP lasted for at least 28 days ,and the expression of GFAP expression in the treatment group was less than that in the control group. Conclusion Methylprednisolone can reduce the expression of GFAP and PCNA in Müller cells of rats’ retinae injured by laser. (Chin J Ocul Fundus Dis, 2002, 18: 299-301)
Objective To study the effects of connective tissue growth factor (CTGF) on retinal Müller cells based on transcriptome analysis of RNA-seq technology.MethodsRetinal Müller cells were divided into the control group and the CTGF treatment group which was continuously cultured with 10 ng/ml of CTGF for 24 h. The influence of CTGF on the migration of Müller cells were tested by scratching experiments. The RNA transcriptome analysis was applied to complete transcriptome sequencing, differentially expressed genes and functional enrichment analysis of the two groups of cells. HiSeq sequencing technology was used to sequence the whole transcriptome of the two groups of cells to obtain biological big data, and analyze the differentially expressed miRNAs on this basis. The functions and signal pathways of differential miRNAs were analyzed through gene annotation (GO) functional significance enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway significant enrichment analysis. Based on transcriptome data, genes with differential expression multiples in the top ten between the two groups were screened out, and the expression of bone morphogenetic protein 4 (BMP4) gene was verified by real time fluorescence quantification PCR (qRT-PCR), immunofluorescence and Western blot.ResultsAfter CTGF stimulation of Müller cells, cell viability and mobility which compared with the control group were significantly increased, with statistically significant differences (t=3.453, P＜0.05). The differential gene expression profile of CTGF induced Müller cells was obtained by RNA transcriptome analysis. Comparing the sequencing results of the two groups, it was found that 325 differentially expressed genes included 152 up-regulated genes and 173 down-regulated genes. The results of GO functional significance enrichment analysis showed that the functions of differential miRNA were mainly divided into three categories: biological processes, cellular components, and molecular functions. These differentially expressed genes were involved in signaling between nervous systems, adhesion between cells, and the interaction between cytokines and their receptors. These differentially expressed genes were involved in different metabolic pathways and biological processes such as tissue inflammation and fibrosis. BMP4 gene was seected for verification through immunofluorescence, qRT-PCR and western blot. The results showed that the expression of BMP4 was significantly higher than that in the control group, and the difference was statistically significant (t=39.490, 10.110, 5.470; P=0.004, 0.001, 0.006).ConclusionCTGF promotes cell proliferation and migration by up-regulating the expression of BMP4 in Müller cells, leading to tissue fibrosis and inducing inflammation.
ObjectiveTo observe the effect of tert-Butylhydroquinone (tBHQ) on the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase (HO)-1 and phosphatidylinositol 3-kinase (PI3K) in high glucose cultured retinal Müller cells; and to investigate the anti-oxidative stress and anti-apoptotic effects of tBHQ.MethodsRetinal Müller cells were divided into normal glucose group (5.5 mmol/L, N group), high glucose group (45 mmol/L, HG group) and tBHQ intervention group (HG+tBHQ group). After retinal Müller cells were cultured with high glucose for 48 hours, the pretreatment with tBHQ (20 μmol/L) induced the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and HO-1. The Müller cells were identified by immunofluorescence staining. The expressions of Nrf2, HO-1, PI3K, B-cell lymphoma-2 (Bcl-2) and Bax were detected by Western blot and real-time fluorescence quantitative PCR. Flow cytometry was used to detect the apoptosis of retinal Müller cells in rats.ResultsMüller cytoplasm and nucleus GS showed strong positive, large cell body, abundant cytoplasm, uniform green fluorescence; nuclear DAPI staining round or oval, clear boundary. The expression of Nrf2 protein (t=4.114, P=0.006), HO-1 protein (t=9.275, P=0.000), Nrf2 mRNA (t=7.292, P=0.000) and HO-1 mRNA (t=15.014, P=0.000) in the HG group were higher than those in the N group. The expressions of Nrf2 protein (t=7.847, P=0.000) ,HO-1 protein (t=7.947, P=0.000), PI3K protein (t=5.397, P=0.002), Bcl-2 protein (t=6.825, P=0.000), Nrf2 mRNA (t=18.046, P=0.000), HO-1 mRNA (t=39.458, P=0.000), PI3K mRNA (t=4.979, P=0.003) and Bcl-2 mRNA (t=9.535, P=0.000) in the HG+tBHQ group were significantly higher than those in the HG group. The protein and mRNA expressions of Bax protein in the HG+tBHQ group were significantly lower than those in the HG group (t=14.998, 16.520; P=0.000, 0.000). Flow cytometry showed that the apoptosis rate of Müller cells in the HG group was significantly higher than that in the N group (t=39.905, P=0.000). The apoptosis rate of Müller cells in the HG+tBHQ group was significantly lower than that in the HG group (t=21.083, P=0.000).ConclusiontBHQ can inhibit the apoptosis of retinal Müller cells by up-regulating the expression of Nrf2, HO-1 and PI3K.
ObjectiveTo observe the protective effect of Zhicao Tea Mixture on Müller cells and the expression of inflammatory factors in mice with diabetic retinopathy.MethodsSeventy-five C57BL/6J mice were randomly divided into the normal control group, diabetes mellitus (DM) group, low concentrations group, medium concentrations group and high concentrations group, with 16 mice in each group. The diabetes model of mice in all groups except the normal control group were established by intraperitoneal injection of STZ (60 mg/kg). Four weeks after the successful modeling, the Zhicao Tea Mixture with low (30 ml/kg), medium (60 ml/kg) and high concentrations (120 ml/kg) were respectively administered by gavage. Weight and blood glucose of mice in each group were measured every two weeks. After 8 weeks, Western blot method was used to detect the mice retina Müller cells activation marker gelatinous fibrous acidic protein (GFAP). Immunofluorescence was performed to detect the expression GFAP and glutamine synthetase (GS). Real-time quantitative PCR (RT-qPCR) and ELISA were used to determine the mRNA and protein expression levels of mouse retinal VEGF, TNF-α, IL-1β and IL-6 respectively.ResultsThe weight of mice in the DM group was lower than that of the normal control group, and the blood glucose was increased. Zhicao Tea Mixture had no effect on the weight of DM mice, but had a significant hypoglycemic effect. The GFAP expression (t=38.318, P＜0.001) in the retina of mice in the DM group was increased and GS expression (t=29.737, P＜0.001) was decreased compared with the control group. The GFAP expression (t=13.677, 19.387, 16.305; P＜0.05) in the retina of mice in the low, medium and high concentrations group were decreased and GS expression (t=5.170, 19.399, 6.705; P＜0.05) were increased compared with the DM group. The expressions of retinal inflammatory factors VEGF, TNF-α, IL-1β and IL-6 in DM group all increased, while the expressions of the above-mentioned inflammatory factors in the retina of mice decreased in the low, medium and high concentrations group.ConclusionZhicao Tea Mixture can decrease the blood glucose of DM mice and reduces the diabetic retinal inflammatory response.