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Several mutant genes for inherited retinal diseases have been identified, but effective treatments are still lacking.The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system can edit human genomic DNA by nonhomologous end joining or homology-directed repair, offering more possibilities for the treatment of hereditary retinal diseases.CRISPR/Cas9 not only can genetically correct patient-derived induced pluripotent stem cells (iPSCs) to observe their differentiation into retinal cells thereby, thereby exploring the pathogenesis of the disease and implementing cell therapy, but can also be delivered to the body via vectors and directly act on target cells to achieve in vivo gene editing.CRISPR/Cas9 gene editing technology in hereditary retinal diseases has been mainly used in retinitis pigmentosa, hereditary X-linked juvenile retinoschisis, and Leber congenital amaurosis 10, of which the in vitro application of CRISPR/Cas9 for Leber congenital amaurosis 10 has entered the clinical trial stage.In this paper, we reviewed the mechanism and key advances of CRISPR/Cas9 and provided an overview of gene editing in IRDs.
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Due to the high incidence and the earlier onset age, high myopia has become an important public health problem in China. Posterior scleral reinforcement surgery has been developed for over 60 years in order to control the rapid progression and complications of high myopia. By suturing a certain size of material on the surface of the posterior eyeball, thickness and elasticity modulus of the local sclera significantly increase. As the result, the rapid growth of the axial length and the chorioretinopathy could be alleviated. At present, controversies about its clinical efficacy and safety still exist, so posterior scleral reinforcement surgery has not been widely carried out all over the world. An in-depth analysis of the mechanism, surgical manipulations and materials, the clinical application status of posterior scleral reinforcement surgery on control of high myopia can provide a basis for further standardized application of this surgery
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At present, tamponade agent which being used in retinal surgery is mainly sterile air, gas and silicone oil. Sterile air is mostly used in the treatment of simple retinal detachment. Gas or silicone oil as tamponade is greatly applied for complicated retinal detachment. In recent years, with the application of micro-invasive vitrectomy under a wide-angle viewing system and perioperative anti-vascular endothelial growth factor drugs, application of intraocular filling materials also has changed. The application of silicone oil is significantly reduced. Percentage rate of gas as tamponade for retinal detachment is reduced. The application of sterile air as tamponade is rising. With selecting indication carefully and picking up the suitable air or gas, doctor will reduce the workload. It will also reduce the social burden and benefit patients.
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Optogenetics is a genetic technique that applies illumination with certain wavelength to modulate the biological activity of cells or subcellular components accurately.This technique is friendly to researchers of ophthalmology due to characteristics of the eyes including transparency, accessibility and comparative independence.Optogenetics can be used in retinal neurons, such as retinal ganglion cells (RGCs), even extended to vascular endothelial cells, immune cells and other ocular cells or cell substructures, which can further our understanding of ocular physiology and provide potential, therapeutic approaches for neurodegenerative diseases, vascular diseases, inflammation and other eye-related diseases.Improvement in effectiveness, safety and comfort is pivotal for this technique to expand application in ophthalmology and for its function to reach the physiology state of nomal eyes.In this review, a comprehensive analysis of optogenetics progress in ophthalmology was performed.Challenges in imaging including light sensitivity, spatial resolution and temporal resolution, and problems in expression involving local and systemic safety, specificity and persistence were reviewed.
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Background Retinal Müller cells are important gliocytcs and the source of retinal stem cells.Researching the biological behavior of Müller cells is of important significance to the study on retinal physiopathological process and stem cell therapy of retinal diseases.To establish a stable culture method of Müller cells is a solid basis of relative basic research.Objective This study was to establish a simple and stable method of isolation and culture of human retinal Müller cells and provide sufficient and high-quality Müller cell source.Methods Human retinal Müller cells were isolated from healthy human donor eyes.The mixture solution of hyaluronidase (100 U) and 0.25％ trypsin were used to digest chopped retinal tissue.The DMEM/F12 medium with 20％ fetal bovine serum (FBS) was added to stop the digestion process.RPMI1640 medium with 20％ FBS was used to culture the cell for 72 hours and then replaced the half medium.The cells were passaged by the RPMI1640 medium with 20％ FBS.The morphology of the cells were examied under the optical microscope,and the expressions of glial fibrillary acidic protein (GFAP),a marker of gliocytes,and glutamine synthetase (GS),a special marker of retinal Müller cells,were detected by immunochemistry and immunofluorescence technology.Results Human retinal Müller cells were successfully isolated by enzyme mixture solution of hyaluronidase (100 U) and O.25％ trypsin.The cells were adherent to walls 24 hours after primary culture and completely merged 9-10 days after culture.The cells showed oval in shape with abundant cytoplasm,and a part of cells presented with cone-shaped bulge bilaterally and ectasia in the posterior containing large nuclei.After cells passage,the cells were enlarged and grew toward polygonal shape.The positive expression of GFAP was observed in more than 95％ cells and strongly positive expression of GS was observed in more than 90％ cells by immunohistochemstry and immunofluorescent staining.Conclusions Human retinal Müller cells can be successfully isolated by hyaluronidase combined with trypsin digestion.Abundent and pure human retinal Müller cells can be obtained by successively using RPMI1640 medium with 20％ FBS and 10％ FBS.
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Background Stem cell transplantation represents a promising treatment option for patients suffering from degenerative disorders.Accumulating evidences indicate that mesenchymal stem cells (MSCs) are able to differentiate into retinal pigment epithelial (RPE)-like cells.However,MSCs are difficult to obtain.Human adipose mesenchymal stem cells (ADSCs) are proved to have similar properties to MSCs,but relevant study is less.Objective This study was to assess the feasibility of human ADSCs differentiating into RPE-like cells and the safety of its application in vivo.Methods The third generation of human ADSCs were incubated into 6-well plate,and 100 ng/ml epithelial growth factor,50 μ mol/L taurine and 5×10-7 mol/L retinoic acid were added into the medium 12 hours after cultured to induce the cells,and conventional cultured cells were used as the control group.Induced cells were traced with PKH26,and Pan-cytoke ratin (Pan-CK) monoclonal antibody was used to identify the cells under the fluorescence microscope.Induced RPE-like cell suspension of 1 μl was intravetreally injected in the right eyes of 6 BALB/c mice,and equal volume of PBS was used in the same way in another 6 mice.The animals were sacrificed 1 month after injection,and the retinal morphology was examined by histopathology under the optical microscope.The ultrastructure of retinal ganglion cells (RGCs) was examined by the transmission electron microscope.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results Cultured human ADSCs grew well with the slender polygone shape.Cell membranes showed the red fluorescence for PKH26 after induced.In addition,Pan-CK was expressed in the cell membranes with the red fluorescence in the induced cells,but the response was absent in the control cells.One month after intravitreal injection,induced cells located on the retinal surface,and the retinal morphology was clear under the optical microscope.No abnormality in RGCs was seen under the transmission electron microscope.Conclusions Human ADSCs can differentiate into RPE-like cells after induction.PKH26 can mark induced cells well.There is no adverse effect of induced cells on retina after intravitreal injection in a short-term duration in mice.
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Recent years have witnessed tremendous progress in vitreoretinal surgery.The treatment of vitreoretinal diseases has increased enormously and its related indications expanded widely with the contribution of the emerging novel technologies,methods,equipment and new ideas.Attaching importance to minimally invasive surgery,application of auxiliary drugs,development of improved equipment and surgical technique were the main features. Further basic and clinical research is necessary to promote innovation and development of vitreoretinal surgery in China to keep pace with and surpass advanced technology.
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Retinal neovascularization is a complicated pathophysiological process as a result of imbalance between angiogenic and anti-angiogenic factors. Correct understanding of the signaling pathways,exploring the critical factors involved in retinal angiogenesis, looking for new strategies by reconstructing the new vessels are helpful for knowing the mechanism of the occurrence and development of reitnal neovascularization, which would be good for preventing and treating retinal neovascularization diseases.
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<p><b>OBJECTIVE</b>To prepare ginkgolide B-loaded self microemulsifying drug delivery system (SMEDDS) and evaluate its quality.</p><p><b>METHOD</b>The solubility of ginkgolide B in different oil, surfactant and co-surfactant were measured by HPLC-ESI-MS. The GB-SMEDDS formulation was optimized by the self emulsifying efficiency of various combinations of oil and mix-surfactant evaluated by using pseudo-temary phase diagram. The preliminary stability of GB-SEMEDDS was evaluated by the variety of loading rate of GB and dispersed medium. The morphology, the particle size and the formulation stability were evaluated after diluting by 0.1 mol x L(-1) HCl.</p><p><b>RESULT</b>The blank self microemulsified system was composed of ethyl oleate-( cremophor EL-lecithin-ethanol, 4: 1:2) (40: 60), the loading dosage was 2.5%. Little influence of GB and emulsified medium was observed on the stability of GB-SEMDDS. After diluted with 0.1 mol x L(-1) HCl, the morphology of the microemulsion was homogeneous small spherical drops observed under electro-microscope. The particle size was (41.6 +/- 1.11) nm, the self microemulsifing time was around 2 min. The formulation was stable within 8 h, without significant changes in particle size and separation of drugs.</p><p><b>CONCLUSION</b>GB-SMEDDS is easy to prepare and its quality is stable. The solubility of GB was significantly improved by SMEDDS.</p>
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Chemistry, Pharmaceutical , Methods , Drug Delivery Systems , Methods , Emulsions , Chemistry , Ginkgolides , Chemistry , Pharmacokinetics , Lactones , Chemistry , Pharmacokinetics , Particle Size , Solubility , Surface-Active Agents , ChemistryABSTRACT
AIM: To investigate the properties of human retinal microvascular endothelial cells (RMECs) at two different age groups. METHODS: Human RMECs with high affinity were isolated from donors at two age groups: 30 d (group A) after birth and 40 - 60 years of age (group B). The RMECs were characterized for expression and localization of endothelial cell markers by immunofluorescence staining of CD31, yon Willebrand factor(vWF) and uptake of acetylated low - density lipoprotein. The ability of tube formation was assessed on Matrigel, and vWF distribution in- cells was ob-served by confocal immunofluorescence microscope and Western blotting analysis, respectively. RESULTS: High purity RMECs can be obtained readily from each group with modified methods. At 6 hours, cells from both groups formed tube structures successfully, but there was a significantly higher incidence of branching in RMECs of infant group (group A) by 27.2%±2.2% compared with adult group (group B) at 12 h (P<0.05). Group A maintained intact structure even at 30h, but group B partially lost the basic tube structure. In addition, vWF was translocated from cytoplasm to nucleus with aging. CONCLUSION: Human RMECs at different ages have specific properties. Cell properties related with age of the donors should be considered in in vitro studies.
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AIM: To observe the effect of ginkgolide B (CB) on the intracellular calcium ion concentration ( [ Ca~(2+) ]_i) and mitochondrial function of cultured rat retinal neurons in vitro. METHODS: in vitro primary culture of rat retinal neurons was used in the experiment. The apoptosis model of glutamate - induced retinal neurons was established and co - cultured with ginkgolide B. The [ Ca~(2+) ]_i and mitochondrial membrane potential of the retinal neurons were detected by laser scanning confocal microscope. RESULTS: Glutamate decreased the survival rate of retinal neurons, increased the apoptosis and the [ Ca~(2+) ]_i, lowered the mitochondrial membrane potential. The [ Ca~(2+) ]_i was clearly diminished and the mitochondrial membrane potential was significantly increased with the GB intervention, and the apoptosis decreased significantly. CONCLUSION: GB protects retinal neurons from glutamate induced neurotoxicity. The effect of GB on retinal neurons might be due to its ability to decrease the [Ca~(2+) ]_i and increase mitochondrial membrane potential.
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BACKGROUND: Embryonic stem cells (ESCs), the seed cells of all mature cells in vivo, are useful tools for nerve transplantation and developmental gene function research. Notch1 signaling pathway is the key pathway to control the ordered neural development and differentiation of many kinds of neural cells, however, there is no report on the dynamic expression of Notch1 signal during the ESC differentiation to date. OBJECTIVE: To investigate the expression of Notch1 protein, transmembrane signal transduction molecule, during directional differentiation of embryonic stem cells into neural cells. DESIGN, TIME AND SETTING: Cell research was carried out between October 2003 and October 2004 at Zhongshan Ophthalmic Center, SUN Yat-sen University, Guangzhou, Guangdong Province, China. MATERIALS: BALB/C mouse embryonic stem cell line Ⅵ (passage 11)was obtained from experimental animal center of SUN Yat-sen University, provided by professor Huang Bing. ESC culture medium was high-glucose DMEM medium with 20% bovine serum and 106 IU/L mouse leukemia inhibitory factor. Induced differentiation medium was high-glucose DMEM medium with 20% fetal bovine.serum and 5×107 mol/L retinoid acid(RA). METHODS: Passage 11 ESCs were resuscitated and incubated by ESC culture medium in incubator at 37℃ with 5% CO2. Passage 11 ESCs were subcultured after 2 or 3 days and RA was added into medium to induce differentiation. Three time points for observation were established: induced for 1, 5 and 9 days. MAIN OUTCOME MEASURES: Morphological changes were observed under inverted phase contrast microscope, MAP-2 antigen expressed in differentiated cells was detected by immunofluorescence method. Immunocytochemistry, Western Blot, flow cytometry assay were used to investigate the Notch1 protein expression. RESULTS: ESCs presented clone-like growth. After induced by RA for 9 days, single neural network was achieved around most of the cell clusters. With the prolongation of induction, MAP-2 positive neural cells increased gradually. Almost all ESC clones expressed Notch1 protein strongly or positively, but Notehl protein expression decreased gradually after induced differentiation (P < 0.01). CONCLUSION: Notch1 signal shuts off progressively during induction of ESCs into neural cells, which suggests Notch1 may play an important role in the differentiation of ESCs into neural cells.
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Objective To evaluate the efficacy and safety of photodynamic therapy(PDT)combined with intravitreaIinjection of bevacizumab for choroidal neovascularization(CNV)caused by agerelated macular degeneration(AMD). Methods A total of 21 eyes of 21 patients with AMD,which was diagnosed by examination of visual acuity,intraocular pressure,ocular fundus,fundus color photography,fundus fluoreseein angiography(FFA),indocyanine green angiography(ICGA)and optic coherence tomography(OCT),were underwent PDT combined with intravitreal injection of Bevacizumab.The patients,15 males(15 eyes)and 6 females(6 eyes),aged from 56 to 78 years,with the average of 68.6years.The best corrected visual acuity:counting fingers/10cm-0.9,logMAR was 1.04±0.41.CNV located in below or side central fovea of macula.There was obvious leakage of fluorescein which examined by FFA and ICGA.The average of retinal thickness of macular foveal was(258.91±78.66)μm.The treatment method of PDT has to according to the way of PDT for TAP and Verteporfin PDT for VIP.Intravitreal infeetion with 1.5mg bevacizumab was performed after three days under surface anesthesia.Follow-up time was 1,3,6,12 months after the treatment. Resuits At last visit,the best corrected visual acuity:counting fingers/10 cm-1.5,logMAR was 1.04±0.41,and the differences are statistically significant compared with before.The BCVA improved four or more lines in 6 eyes(28.57%),improved two to four lines in 9 eyes(42.86%),stabilized(±1 line or no change)in 6 eyes(28.57%)and decreased in none.The average intraocular pressure was(15.20±2.41)mmHg after surgery,and the differences was not statistically significant compared with before(P＞0.05).FFA and lCGA showed CNV complete closure in 13 eyes(61.90%).partial closure in 8 eyes(38.10%).The average of retinal thickness of macular foveal was(127.38±20.14)μm(P＜0.01). Conclusion Combining treatment with PDT and intravitreal injection of Bevacizumab is safe and effective for CNV which caused by AMD.It has significant improvement in BCVA.1eakage of CNV and retinal edema.
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OBJECTIVE:To observe the protective effect of the traditional Chinese medicine Yiqimingmu oral solution on photochemical damage of retina in rats. METHODS:SD rats were divided into four groups randomly:negative control group, model group, low dose Yiqimingmu oral solution group and high dose Yiqimingmu oral solution group. All the groups except the control one were continually exposed to Lux (1 900?106.9) green fluorescent light for 24 hours to establish retinal photochemical damage model. The low dose and high dose groups were administered intragastrically with Yiqimingmu oral solution (15mL?kg-1?d-1 and 30mL?kg-1?d-1) 7 days before light expose. Normal saline solution was administered intragastrically in control and model group. At 6 hours and 6, 14 days after light expose, the content of malandialdehyde (MDA) and activity of SOD in rats' retina were measured. The histopathologic changes were observed by transmission electron microscopy and light microscopy. RESULTS:At 6 and 14 days after light expose, the activity of SOD in Yiqimingmu solution high dose group were higher than in the model control group(P
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Objective To observe the changes of VEGF and Ang-2 expression in streptozotocin(STZ)-induced diabetic rat retina after insulin therapy and explore possible roles of insulin in the development of diabetic retinopathy. Methods Diabetes was induced in 8-week-old male wistar rats by a single intraperitoneal injection of STZ. After three weeks,animals were randomly divided into four groups:(1)diabetic rats received intensive insulin therapy for 20 days;(2)diabetic rats received unregular insulin therapy,which caused the abrupt fluctuation of glycemic level;(3)diabetic control rats; and (4)normal control rats. After treatment,the animals were sacrificed with an overdose of anesthesia,and the eyes were enucleated and fixed in 4% paraformaldehyde immediately. Paraffin sections of retina were prepared.Expression of VEGF and Ang-2 was assessed by immunofluorescence stain and images analysis. Results Quantitative analysis showed VEGF and Ang-2 protein expression was increased by 2.38-fold and 2.41-fold in diabetic rats retinas as compared to non-diabetic rats retinas respectively (P0.05). Conclusion Intensive insulin therapy could decrease VEGF and Ang-2 expression in retina and has protective effect on diabetic retinopathy in STZ-diabetic rats.
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Objective To observe the adhension and stracking of leukocyte in the capillary vessels, and investigate the relationship between leukocyte and microvascular morphologic changes in retinal microvessels of rats with early diabetes. Methods A total of 90 healthy adult male Wistar rats were randomly divided into control and diabetes (induced by Streptozotocin, STZ) groups with 45 rats in each group. The rats in the diabetic group were further divided into 3, 7, and 14 days groups with 5 rats in each group, and 30, 90, and 180 days groups with 10 rats in each group. The right eyes of rats in each group were prepared for retinal digest preparations. The expression of leukocyte common antigen (CD45) was detected by immunohistochemical staining. Results Few CD45 positive cells in the retinal capillaries were seen in the control group. The expression of CD45 was significantly increased in the retinal capillaries 3 days after diabetes induction, and reached a peak at the 14th day. Morphological changes including capillary telangiectasia, atresia, and irregularity of capillary caliber were found in the retinal capillaries of rats 90 days after diabetes induction. The changes were aggravated 180 days after diabetes induction. Conclusion Leukocyte adhesion occurs in the early stage of diabetic retinopathy (DR), and is the beginning of the microvascular pathological changes. Leukocyte adhesion may play an important role in the occurrence and development of DR as the foundation of microvascular morphological changes.
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Objective To observe the ultrastructural characteristics of human retinal progenitor cells cultured in vitro. Methods Six 5-month-old human fetuses(12 eyes)without eye diseases were selected. Retinal progenitor cells from the retina of one eye of each fetus were cultured in vitro,and observed by transmission electronic microscopy(TEM); while those from the other eye were directly observed by TEM. Results Abundant heterochromatin were found in the karyon of 5-month embryonic retinal neuroepithelial cells,and the figure of the karyons was irregular.A few scattered initial cells were seen in retinal neuroepithelial layer with large karyon,smooth surface,abundant euchromatin,and distinct nucleolus.The human retinal progenitor cells cultured in vitro had the same ultrastructural characteristics as the initial cells:with huge karyon which almost occupied the whole cell,little cytoplasm,distint nucleolus,abundant euchromatin,and little heterochromatin.The cells clung to each other in the neural globoid cell mass.The size of the outer cells was large,and karyokinesis could be found. Conclusion The cultured human retinal progenitor cells are provided with the same ultrastructure characteristics as the initial cells.
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Objective To evaluate the indications, effectiveness and complications of vitreoretinal surgery using the 25G transconjunctival sutureless vitrectomy system (TSV25G) under the topical anesthesia. Methods The clinical and follow-up data of 22 eyes of 22 patients undergone vitreo-retinal surgery using TSV25G under the topical anesthesia were retrospectively analyzed. All of the patients were monocular sickened, including idiopathic macular hole in 10 eyes, idiopathic macular pucker in 6, vitreoretinal traction syndrome in 4, and vitreous hemorrhage associated with branch retinal vein occlusion in 2. Peeling of epiretinal membrane and/or internal limiting membrane, intraocular laser coagulation, air-fluid exchange and tamponiding of C 3F 8 were performed according to the condition of diseases. The postoperative follow-up was 1-11 months, with the mean duration of 6.4 months. The effect of analgesia, cooperation with the patients, operative effect and complications in and after the surgery were observed. Results The operations finished successfully in all of the eyes under the topical anesthesia. The operation duration ranged from 20 to 25 minutes with average of 22 minutes. The patients cooperated with the doctor well without any discomfort. Two days after the surgery, edema of the wounded conjunctiva was found, and recovered 7 days later. A light pigment dot on the surface of the sclera could be seen at the first month. The complications included transient increasing of intraocular pressure in 2 eyes, feather-like opacity of lens in 5 eyes, vitreous hemorrhage in 1 eye, and air-bleb under conjunctiva in 2 eyes. No other complications related with the cut were found. The macular hole closed in 9 eyes with idiopathic macular hole, and the other 1 had the smaller but not closed hole. Idiopathic macular pucker, vitreoretinal traction syndrome, and vitreous hemorrhage associated with branch retinal vein occlusion were cured successfully. Conclusions Vitreoretinal surgery using the TSV25G under the topical anesthesia has many advantages such as simple procedure, short operation time, micro-invasion, less complications and rapid revovery, and mainly serves simple manipulation in some simple diseases such as idiopathic macular hole, vitreo-retinal traction syndrome, and simple hemorrhage.
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AIM:To observe the effect of ginkgolide B (GB) on the intracellular calcium ion concentration ([Ca2+]i) and mitochondrial function of cultured rat retinal neurons in vitro.METHODS:in vitro primary culture of rat retinal neurons was used in the experiment. The apoptosis model of glutamate-induced retinal neurons was established and co-cultured with ginkgolide B. The [Ca2+]i and mitochondrial membrane potential of the retinal neurons were detected by laser scanning confocal microscope.RESULTS:Glutamate decreased the survival rate of retinal neurons,increased the apoptosis and the [Ca2+]i,lowered the mitochondrial membrane potential. The [Ca2+]i was clearly diminished and the mitochondrial membrane potential was significantly increased with the GB intervention,and the apoptosis decreased significantly.CONCLUSION:GB protects retinal neurons from glutamate induced neurotoxicity. The effect of GB on retinal neurons might be due to its ability to decrease the [Ca2+]i and increase mitochondrial membrane potential.
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AIM: To investigate the feasibility and effect of directly differentiation of embryonic stem cells(ESC) into neural cells induced by retinoid acid(RA) without embryonic body(EB) culture period in vitro.METHODS: ESC were digested and divided into 4 groups: group A and B were undergone EB culturing.After that,cells in group A were induced by RA,cells in group B were differentiated spontaneously,cells in group C were committedly induced by RA directly without EB culturing,and cells in group D were differentiated spontaneously without EB period.Morphologic changes were observed under inverted microscope and scanning electron microscope.MAP-2 and GFAP were detected by immunocytochemistry and flow cytometry after differentiated for 9 days.RESULTS: In groups A or C,neuron-like cells increased gradually,forming neural network.At the 9th day,a large part of cells in these groups were MAP-2 positive cells,and the positive rate was higher than that in groups B or D(P0.05).CONCLUSION: ESC was directly induced into neural cells by RA without EB culture period in vitro.This modified method has the same effect as the traditional RA 4-/4+ assay and can replace the traditional method.