Chinese Guideline on the Management of Polypoidal Choroidal Vasculopathy (2022).

Background In mainland China, patients with neovascular age-related macular degeneration (nAMD) have approximately an 40% prevalence of polypoidal choroidal vasculopathy (PCV). This disease leads to recurrent retinal pigment epithelium detachment (PED), extensive subretinal or vitreous hemorrhages, and severe vision loss. China has introduced various treatment modalities in the past years and gained comprehensive experience in treating PCV.Methods A total of 14 retinal specialists nationwide with expertise in PCV were empaneled to prioritize six questions and address their corresponding outcomes, regarding opinions on inactive PCV, choices of anti-vascular endothelial growth factor (anti-VEGF) monotherapy, photodynamic therapy (PDT) monotherapy or combined therapy, patients with persistent subretinal fluid (SRF) or intraretinal fluid (IRF) after loading dose anti-VEGF, and patients with massive subretinal hemorrhage. An evidence synthesis team conducted systematic reviews, which informed the recommendations that address these questions. This guideline used the GRADE (Grading of Recommendations, Assessment, Development, and Evaluation) approach to assess the certainty of evidence and grade the strengths of recommendations. Results The panel proposed the following six conditional recommendations regarding treatment choices. (1) For patients with inactive PCV, we suggest observation over treatment. (2) For treatment-na?ve PCV patients, we suggest either anti-VEGF monotherapy or combined anti-VEGF and PDT rather than PDT monotherapy. (3) For patients with PCV who plan to initiate combined anti-VEGF and PDT treatment, we suggest later/rescue PDT over initiate PDT. (4) For PCV patients who plan to initiate anti-VEGF monotherapy, we suggest the treat and extend (T&E) regimen rather than the pro re nata (PRN) regimen following three monthly loading doses. (5) For patients with persistent SRF or IRF on optical coherence tomography (OCT) after three monthly anti-VEGF treatments, we suggest proceeding with anti-VEGF treatment rather than observation. (6) For PCV patients with massive subretinal hemorrhage (equal to or more than four optic disc areas) involving the central macula, we suggest surgery (vitrectomy in combination with tissue-plasminogen activator (tPA) intraocular injection and gas tamponade) rather than anti-VEGF monotherapy. Conclusions Six evidence-based recommendations support optimal care for PCV patients' management.

Tissue-Specific Gamma-Flicker Light Noninvasively Ameliorates Retinal Aging.

Aging is a risk factor for multiple retinal degeneration diseases. Entraining brain gamma oscillations with gamma-flicker light (γFL) has been confirmed to coordinate pathological changes in several Alzheimer's disease mouse models and aged mice. However, the direct effect of γFL on retinal aging remains unknown. We assessed retinal senescence-associated beta-galactosidase (ß-gal) and autofluorescence in 20-month-old mice and found reduced ß-gal-positive cells in the inner retina and diminished lipofuscin accumulation around retinal vessels after 6 days of γFL. In immunofluorescence, γFL was further demonstrated to ameliorate aging-related retinal changes, including a decline in microtubule-associated protein 1 light chain 3 beta expression, an increase in complement C3 activity, and an imbalance between the anti-oxidant factor catalase and pro-oxidant factor carboxymethyl lysine. Moreover, we found that γFL can increase the expression of activating transcription factor 4 (ATF4) in the inner retina, while revealing a decrease of ATF4 expression in the inner retina and positive expression in the outer segment of photoreceptor and RPE layer for aged mice. Western blotting was then used to confirm the immunofluorescence results. After mRNA sequencing (NCBI Sequence Read Archive database: PRJNA748184), we found several main mechanistic clues, including mitochondrial function and chaperone-mediated protein folding. Furthermore, we extended γFL to aged Apoe-/- mice and showed that 1-m γFL treatment even improved the structures of retinal-pigment-epithelium basal infolding and Bruch's membrane. Overall, γFL can orchestrate various pathological characteristics of retinal aging in mice and might be a noninvasive, convenient, and tissue-specific therapeutic strategy for retinal aging.

Gut microbiota induced abnormal amino acids and their correlation with diabetic retinopathy.

AIM: To explore the correlation of gut microbiota and the metabolites with the progression of diabetic retinopathy (DR) and provide a novel strategy to elucidate the pathological mechanism of DR. METHODS: The fecal samples from 32 type 2 diabetes patients with proliferative retinopathy (PDR), 23 with non-proliferative retinopathy (NPDR), 27 without retinopathy (DM), and 29 from the sex-, age- and BMI- matched healthy controls (29 HC) were analyzed by 16S rDNA gene sequencing. Sixty fecal samples from PDR, DM, and HC groups were assayed by untargeted metabolomics. Fecal metabolites were measured using liquid chromatography-mass spectrometry (LC-MS) analysis. Associations between gut microbiota and fecal metabolites were analyzed. RESULTS: A cluster of 2 microbiome and 12 metabolites accompanied with the severity of DR, and the close correlation of the disease progression with PDR-related microbiome and metabolites were found. To be specific, the structure of gut microbiota differed in four groups. Diversity and richness of gut microbiota were significantly lower in PDR and NPDR groups, than those in DM and HC groups. A cluster of microbiome enriched in PDR group, including Pseudomonas, Ruminococcaceae-UCG-002, Ruminococcaceae-UCG-005, Christensenellaceae-R-7, was observed. Functional analysis showed that the glucose and nicotinate degradations were significantly higher in PDR group than those in HC group. Arginine, serine, ornithine, and arachidonic acid were significantly enriched in PDR group, while proline was enriched in HC group. Functional analysis illustrated that arginine biosynthesis, lysine degradation, histidine catabolism, central carbon catabolism in cancer, D-arginine and D-ornithine catabolism were elevated in PDR group. Correlation analysis revealed that Ruminococcaceae-UCG-002 and Christensenellaceae-R-7 were positively associated with L-arginine, ornithine levels in fecal samples. CONCLUSION: This study elaborates the different microbiota structure in the gut from four groups. The relative abundance of Ruminococcaceae-UCG-002 and Parabacteroides are associated with the severity of DR. Amino acid and fatty acid catabolism is especially disordered in PDR group. This may help provide a novel diagnostic parameter for DR, especially PDR.

Phosphomannopentaose sulfate (PI-88) suppresses angiogenesis by downregulating heparanase and vascular endothelial growth factor in an oxygen-induced retinal neovascularization animal model.

PURPOSE: Vascular endothelial growth factor (VEGF) is the most potent angiogenic mitogen, and has been associated with angiogenesis. Heparanase is an endoglycosidase that specifically cleaves heparan sulfate side chains, which can induce VEGF expression. The aims of the present study were to evaluate the heparanase expression and its relationship with VEGF in the retina of oxygen-induced retinopathy (OIR) mice, and to investigate the effect of the heparanase inhibitor phosphomannopentaose sulfate (PI-88) in the OIR retinas. METHODS: Seventy-seven newborn C57BL/6 mice were involved in this study. On postnatal day 7 (P7), pups were exposed to a hyperoxia condition (75% oxygen) for 5 days, and on P12, the mice were returned to room air. Control mice were exposed to room air from birth until P17, with normally developing retinal vasculature. PI-88 was administered intraperitoneally to OIR mice at a dose of 35.7 mg/kg/day for 5 consecutive days. The expression level of heparanase and VEGF in the retinas was assayed using immunohistochemistry, Q-RT-PCR, and western blot. RESULTS: The expression levels of heparanase and VEGF were increased in the OIR retinas compared with the control mice. The Q-RT-PCR results showed that the mRNA expression levels of heparanase and VEGF in OIR retina were increased 1.71 fold (p<0.0001) and 4.34 fold (p<0.0001), respectively. The western blot results showed that the protein expression levels of heparanase and VEGF were increased 1.49 fold (p<0.0001) and 1.72 fold (p<0.0001), respectively, in the OIR retinas compared with the normal retinas. The immunohistochemistry analysis revealed that the heparanase and VEGF signals were intense in the retinal vascular endothelia of the OIR mice but faint in those of the normal controls. The increased protein and mRNA expression levels of heparanase and VEGF in the mouse retinas were significantly decreased by PI-88 administration (p<0.0001). CONCLUSIONS: Heparanase expression was upregulated and correlated with an increase in VEGF expression in the OIR mouse retinas, and might be involved in the progress of retinopathy of prematurity. Inhibition of heparanase expression by PI-88 could be used as a novel therapeutic method for retinopathy of prematurity.

[Relationship between gender and posterior pole choroidal thickness in normal eyes].

OBJECTIVE: To investigate and compare the choroidal thickness between healthy male and female subjects. METHOD: Six-hundred and twenty eyes of 310 healthy volunteers with no ophthalmic disease history were recruited, including 152 males and 158 females. All volunteers were subgrouped into Group A to F according to their ages. Enhanced depth imaging choroidal scans were obtained in all eyes by using spectral-domain optical coherence tomography. Subfoveal choroidal thickness (SFCT) and choroidal thickness at 1 mm/3 mm superior, inferior, nasal and temporal to the fovea were measured. Choroidal thickness was compared between male and female in the subgroups with different age. RESULTS: Mean SFCT was higher in 152 males (298.02 ± 101.47) µm than that in 158 females (256.28 ± 90.87) µm with statistically significant difference (t' = 4.853, P < 0.05). Choroid at 1 mm and 3 mm from the fovea were also thicker in the male (t' = 5.050, t = 4.597, t = 5.225, t = 5.363, t = 5.608, t' = 4.239, t = 4.108, t' = 5.589; P < 0.05). In any subgroup from A to E, SFCT in male was significantly thicker than female, after adjusted for refractive error (t = 2.343, t' = 2.163, t = 3.239, t = 2.181, t' = 2.982; P < 0.05). In Group F, mean SFCT in male was thicker than female, but without statistical significance (t' = 0.681, P > 0.05). CONCLUSIONS: Gender was one of the factors that affect the choroid thickness in healthy populations. In subjects under 70, male have thicker choroid than female. This result at least partially explained the gender predilection of macular diseases, such as central serous chorioretinopathy and idiopathic macular hole.

Lutein delays photoreceptor degeneration in a mouse model of retinitis pigmentosa.

Retinitis pigmentosa is a retinal disease characterized by photoreceptor degeneration. There is currently no effective treatment for retinitis pigmentosa. Although a mixture of lutein and other antioxidant agents has shown promising effects in protecting the retina from degeneration, the role of lutein alone remains unclear. In this study, we administered intragastric lutein to Pde6brd10 model mice, which display degeneration of retinal photoreceptors, on postnatal days 17 (P17) to P25, when rod apoptosis reaches peak. Lutein at the optimal protective dose of 200 mg/kg promoted the survival of photoreceptors compared with vehicle control. Lutein increased rhodopsin expression in rod cells and opsin expression in cone cells, in line with an increased survival rate of photoreceptors. Functionally, lutein improved visual behavior, visual acuity, and retinal electroretinogram responses in Pde6brd10 mice. Mechanistically, lutein reduced the expression of glial fibrillary acidic protein in Müller glial cells. The results of this study confirm the ability of lutein to postpone photoreceptor degeneration by reducing reactive gliosis of Müller cells in the retina and exerting anti-inflammatory effects. This study was approved by the Laboratory Animal Ethics Committee of Jinan University (approval No. LACUC-20181217-02) on December 17, 2018.

[The effect of rAAV2-PEDF on proliferation of human retinal capillary endothelium cells].

OBJECTIVE: To culture human retinal capillary endothelium cells (HRCECs) in vitro and explore the effect of rAAV2-PEDF on proliferation of HRCEs. METHODS: Retinas were digested by 2.5% trypsin and 0.1% collagenase I in order. The isolated cells were cultured on fibronectin-coated dishes in media of human endothelial-sFM basal growth medium (HE-SFM BGM) with 10% fetal bovine serum, insulin-transferrin-selenium (ITS) and endothelial cell growth factor (ECGF). The cultured cells were identified by anti-factor VIII related antigen though immunohistochemistry stain. The effect of hypoxia induced by CoCl2 on proliferation of HRCECs was assessed by MTT assay. After rAAV2-PEDF were transfected into HRCECs, the EGPF positive cells were observed by laser confocal scanning microscopy, the protein expression of PEDF were detected by Western blot, and the proliferation of HRCECs were checked by MTT assay. Flow cytometry was used to analyze the apoptosis of HRCECs. RESULTS: Cultured HRCECs attached in the bottom of dishes in 48 h - 72 h and grew to confluence in 2 weeks after seeding. HRCECs were with a positive brown staining for factor VIII. EGPF positive cells were seen under laser confocal scanning microscopy after 48 h of rAAV2-EGFP transfection. The expression level of PEDF protein was higher in experimental group than in control group. The results of MTT assay showed the numeric value OA was 0.085 ± 0.021 in normal group and 0.166 ± 0.024 in hypoxia group (t = 3.938, P < 0.05). In normal oxygen condition, the numeric value OA was 0.171 ± 0.011 in normal control group, 0.178 ± 0.016 in rAAV2-EGFP treated group, and 0.169 ± 0.017 in rAAV2-PEDF treated group (F = 0.01, P > 0.05). In hypoxia condition, the numeric value OA was 0.166 ± 0.013 in CoCl(2) treated group, 0.155 ± 0.012 in CoCl(2) + rAAV2-EGFP treated group, and 0.116 ± 0.015 in CoCl(2) + rAAV2-PEDF treated group. In normal oxygen condition, the ratio of apoptosis was 2.3% in normal control group, and 3.3% in rAAV2-EGFP treated group, and 1.7% in rAAV2-PEDF treated group. In hypoxia condition, the ratio of apoptosis was 3.6% in CoCl(2) treated group, 6.7% in CoCl(2) + rAAV2-EGFP treated group, and 36.4% in CoCl(2) + rAAV2-PEDF treated group. CONCLUSIONS: PEDF gene can stably express in HRCECs after rAAV2-PEDF transfection and can obviously inhibit proliferation of HRCECs in hypoxia.

[Differential expression and significance of complement C4b and transthyretin in proliferative vitreoretinopathy].

OBJECTIVE: To investigate the differential expression of complement C4b and transthyretin in proliferative vitreoretinopathy (PVR). METHODS: It was a controlled experimental study. Human vitreous samples of 5 patients with PVR were analyzed by using two-dimensional gel electrophoresis and mass spectrometry, and the results were compared with those from normal control vitreous obtained from donor eyes. An in vivo model of PVR was created by intravitreous injection of cultured rabbit retinal pigment epithelial (RPE) cells. The vitreous of PVR models were analyzed by enzyme linked immunosorbent assay (ELISA) to confirm the proteomic results from the PVR patients. RESULTS: Seventy nine various proteins were expressed differently between PVR and normal vitreous, among which nine up-regulated proteins including complement C4b, transthyretin (TTR), and 7 albumins were identified by mass spectrometry. The up-regulation of complement C4b and TTR in PVR patients was also confirmed by ELISA. The concentration of complement C4b and TTR in normal vitreous were (20.18 ± 1.97) mg/L and (88.58 ± 8.84) mg/L respectively, in PVR patients were (38.1 ± 5.79) mg/L and (112.57 ± 6.89) mg/L respectively, difference significantly between these two groups (C4b: t = 11.54, TTR:t = 9.24; P < 0.05). CONCLUSIONS: Differences of complement C4b and TTR expression were observed between PVR and normal vitreous. These results have lead to the assumption that there is a connection between elevated concentrations of both complement C4b and TTR and the pathogenesis of PVR and further studies on the functions of these proteins are required.

Characteristics of neural growth and cryopreservation of the dorsal root ganglion using three-dimensional collagen hydrogel culture versus conventional culture.

In vertebrates, most somatosensory pathways begin with the activation of dorsal root ganglion (DRG) neurons. The development of an appropriate DRG culture method is a prerequisite for establishing in vitro peripheral nerve disease models and for screening therapeutic drugs. In this study, we compared the changes in morphology, molecular biology, and transcriptomics of chicken embryo DRG cultured on tissue culture plates (T-DRG) versus three-dimensional collagen hydrogels (C-DRG). Our results showed that after 7 days of culture, the transcriptomics of T-DRG and C-DRG were quite different. The upregulated genes in C-DRG were mainly related to neurogenesis, axon guidance, and synaptic plasticity, whereas the downregulated genes in C-DRG were mainly related to cell proliferation and cell division. In addition, the genes related to cycles/pathways such as the synaptic vesicle cycle, cyclic adenosine monophosphate signaling pathway, and calcium signaling pathway were activated, while those related to cell-cycle pathways were downregulated. Furthermore, neurogenesis- and myelination-related genes were highly expressed in C-DRG, while epithelial-mesenchymal transition-, apoptosis-, and cell division-related genes were suppressed. Morphological results indicated that the numbers of branches, junctions, and end-point voxels per C-DRG were significantly greater than those per T-DRG. Furthermore, cells were scattered in T-DRG and more concentrated in C-DRG, with a higher ratio of 5-ethynyl-2'-deoxyuridine (EdU)-positive cells in T-DRG compared with C-DRG. C-DRG also had higher S100 calcium-binding protein B (S100B) and lower α-smooth muscle actin (α-SMA) expression than T-DRG, and contained fewer terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells after 48 hours of serum starvation. After cryopreservation, C-DRG maintained more intact morphological characteristics, and had higher viability and less TUNEL-positive cells than T-DRG. Furthermore, newly formed nerve bundles were able to grow along the existing Schwann cells in C-DRG. These results suggest that C-DRG may be a promising in vitro culture model, with better nerve growth and anti-apoptotic ability, quiescent Schwann cells, and higher viability. Results from this study provide a reference for the construction, storage, and transportation of tissue-engineered nerves. The study was approved by the Ethics Committee of Aier School of Ophthalmology, Central South University, China (approval No. 2020-IRB16), on March 15, 2020.

Overexpression of nuclear transport factor 2 may protect against diabetic retinopathy.

PURPOSE: We performed human, animal, and in vitro studies to examine the potential role of nuclear transport factor 2 (NTF2) in conferring resistance to diabetic retinopathy (DR). METHODS: Blood NTF2 levels were assessed in two groups of patients with type 2 diabetes mellitus. Group P patients had a history of proliferative DR (PDR), while group N patients did not. The retinal vasculature was examined in diabetic rats three months after they received an intravitreal injection of a recombinant adeno-associated virus (rAAV) vector overexpressing NTF2 (rAAV2-NTF2). Control rats were treated with rAAV2 only. Rat retinal capillary endothelial cells (RRCECs) were infected with rAAV2-NTF2, or with a vector expressing siRNA targeted against NTF2, to assess the effects of overexpression and inhibition of NTF2 on vascular endothelial growth factor (VEGF) expression (mRNA and protein). RESULTS: There was a strong trend for patients with DR to have lower blood NTF2 levels compared to those who did not have DR (0.10+/-0.01 versus 0.20+/-0.08, p=0.079). There was significantly less retinal blood vessel leakage in diabetic rats infected with rAAV2-NTF2 compared to controls (16.5+/-2.9 versus 24.7+/-7.3, p=0.039). These rats exhibited normal retinal vasculature and blood-retinal barrier function. VEGF expression was inhibited by NTF2 overexpression and stimulated by NTF2 inhibition, (protein [0.41+/-0.05 versus 0.23+/-0.06] and mRNA [0.37+/-0.04 versus 0.23+/-0.06] p<0.01 for all). CONCLUSIONS: These finding suggest that NTF2 is a potential mediator of retinal vasculature integrity. NTF2 may act by altering VEGF expression, thereby influencing the development of DR in patients with diabetes mellitus.

[Effect of huoxueyiqimingmu capsule on retinal degenerative diseases].

OBJECTIVE: To investigate therapeutic effects of Huoxueyiqimingmu capsule on retinal degenerative diseases. METHODS: Sixty SD rats were randomly divided into three groups: negative control group, light damage model group, Huoxueyiqimingmu capsule group. The light damage model was used as retinal degenerative diseases animal model. Normal saline and Huoxueyiqimingmu capsule were respectively administrated after light damage. After 14 days post-treatment, the change of electroretinogram was observed, content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) in rats retina were determined, and histopathologic changes were observed with transmission electron microscopy and light microscopy. RESULTS: After 14 days post-treatment, Huoxueyiqimingmu capsule had protective effects on fall of ERG a wave and b wave amplitude induced by light damage. The activity of SOD in Huoxueyiqimingmu capsule group was higher than that in light damage model group (P < 0.01), while the content of MDA in Huoxueyiqimingmu capsule group was lower than that in light damage model group (P < 0.01). The histopathologic injury of rat retina in Huoxueyiqimingmu capsule group was lighter than that in light damage model group by means of transmission electron microscopy and light microscopy. CONCLUSIONS: Huoxueyiqimingmu capsule is effective to treat retinal degenerative diseases.

Differentiation of retinal ganglion cells from induced pluripotent stem cells: a review.

Glaucoma is a common optic neuropathy that is characterized by the progressive degeneration of axons and the loss of retinal ganglion cells (RGCs). Glaucoma is one of the leading causes of irreversible blindness worldwide. Current glaucoma treatments only slow the progression of RGCs loss. Induced pluripotent stem cells (iPSCs) are capable of differentiating into all three germ layer cell lineages. iPSCs can be patient-specific, making iPSC-derived RGCs a promising candidate for cell replacement. In this review, we focus on discussing the detailed approaches used to differentiate iPSCs into RGCs.

5-aza-2'-deoxycytidine in the regulation of antioxidant enzymes in retinal endothelial cells and rat diabetic retina.

AIM: To investigate the roles of a DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) in the regulation of antioxidant enzymes in diabetic retinopathy (DR) models. METHODS: DNMTs expressions and activity, and changes of two key antioxidant enzymes in DR, MnSOD (encoded by SOD2 gene) and glutathione S-transferase theta 1 (GSTT1), were quantified in the isolated human retinal endothelial cells (HRECs) exposed to high glucose (HG) with or without 5-aza-dC treatment. The downstream exacerbating factors including vascular endothelial growth factor (VEGF), intercellular adhesion molecule 1 (ICAM-1) and matrix metalloproteinase 2 (MMP2), which are implicated in the pathogenesis of DR and closely related to oxidative stress were also analyzed. The key parameters were confirmed in the retina from streptozotocin (STZ) diabetic rats. RESULTS: DNMTs expression and DNMT activity was induced in HRECs exposed to HG. Hyperglycemia decreased MnSOD and GSTT1 expression. 5-aza-dC administration effectively suppressed DNMTs expression and activity and reversed the MnSOD and GSTT1 expression under HG condition. VEGF, ICAM-1 and MMP2 induced by HG were also suppressed by 5-aza-dC treatment. Similar results were observed in the retina from STZ diabetic rats. CONCLUSION: Our findings suggest that DNA methylation may serves as one of the mechanisms of antioxidant defense system disruption in DR progression. Modulation of DNA methylation using pharmaceutic means such as DNMT inhibitors could help maintain redox homeostasis and prevent further progression of DR.

Scutellarein inhibits hypoxia- and moderately-high glucose-induced proliferation and VEGF expression in human retinal endothelial cells.

AIM: This study was designed to examine the effect of scutellarein on high glucose- and hypoxia-stimulated proliferation of human retinal endothelial cells (HREC). METHODS: HREC were cultured under normal glucose (NG), moderate, and high glucose (NG supplemented with 10 or 25 mmol/L D-glucose) and/or hypoxic (cobalt chloride treated) conditions. Cell proliferation was evaluated by a cell counting kit. The expression of vascular endothelial growth factor (VEGF) was assessed by Western blot analysis. RESULTS: The proliferation of HREC was significantly elevated in response to moderately-high glucose and hypoxic conditions. The combination of high glucose and hypoxia did not have any additive effects on cell proliferation. Consistent with the proliferation data, the expression of VEGF was also upregulated under both moderately-high glucose and hypoxic conditions. The treatment with scutellarein (1x10(-11) to 1x10(-5) mol/L) significantly inhibited high glucose- or hypoxia-induced cell proliferation and VEGF expression. CONCLUSION: Both hypoxia and moderately-high glucose were potent stimuli for cell proliferation and VEGF expression in HREC without any significant additive effects. Scutellarein is capable of inhibiting the proliferation of HREC, which is possibly related to its ability to suppress the VEGF expression.

[Experimental studies on the existence and distribution characteristics of neural stem cells in adult human ciliary body and retina].

OBJECTIVE: To investigate the existence and distribution characteristics of neural stem cells in the eyes of adult human ciliary body and retina. METHODS: Eight eyes from 20 - 40 years old health adult and 3 infant eyes were obtained from Guangdong Eye Bank and were used in the present studies. The protein and mRNA expressions of neural progenitor cell-specific antigen nestin in the ciliary body and retina were detected by immunohistochemical staining and reverse RT-PCR assays, respectively. The ultrastructural and distribution characteristics of these cells were studied by transmission electron microscopy (TEM). RESULTS: Immunohistochemical staining showed that there were a few nestin-positive cells existed in pigment epithelium layer of ciliary body and inner nuclear layer (INL) of retina. mRNA expression of nestin gene was detected in both the ciliary body and retina of the infant and adult eyes by RT-PCR assays. The expression of nestin in the infant ciliary body was higher than that of the adult eye. By TEM, a few cells with the characteristic morphology of neural stem cells were identified and their location was consistent with the results of immunohistochemical staining. CONCLUSION: Cells with the features of neural stem cells are present in the pigment epithelium layer of ciliary body and the inner nuclear layer of retina in adult human eyes.

[Changes of tight junction protein and GFAP in the retina of experimental diabetic rats and their relationship with blood aqueous barrier].

OBJECTIVE: To investigate the changes of vascular endothelial cell tight junction protein (occludin) and glial cell morphology as well as their relationship with blood-retinal barrier (BRB) in the retina of diabetic rats. METHODS: The distribution of occludin and GFAP were explored by immunofluorescence histochemical studies in the retina of streptozotocin (STZ)-diabetic rats and age-matched control rats. Evans blue was used to evaluate the impairment of BRB. RESULTS: GFAP immunoreactivity was limited to ganglion cell layer and nerve fiber layer in the control retina. GFAP immunoreactivity was significantly increased in ganglion cell layer and nerve fiber layer in one month diabetic rats. GFAP positive Müller cells were increased in three months and six months diabetic rats. Occludin immunoreactivity progressively decreased in three months and six months diabetic rats but not in the one month diabetic rats. Evans blue injection showed a progressive impairment of BRB. CONCLUSIONS: Astrocytes activation in the early stage of diabetes plays an important role in the maintaining of the BRB function. But the activation of Müller cells in the later stage destroyed the BRB eventually. These changes are consistent with the concept that BRB changes caused by altered glial-endothelial cell interactions contributes to the occurrence of diabetic retinopathy.

[A preliminary study on intravitreal injection of bevacizumab for treatment of neovascular age-related macular degeneration].

OBJECTIVE: To evaluate the short-term safety of intravitreous bevacizumab (Avastin) and its effects on visual acuity (VA) and subfoveal choroidal neovascularization (CNV) in patients with neovascular age-related macular degeneration (ARMD). METHODS: Single-center, uncontrolled clinical study. Five ARMD patients (5 eyes) with subfoveal CNV and best-corrected VA (BCVA) less than 0.1 were participated. Patients were treated with an intravitreous injection of bevacizumab (1.5 mg, 0.06 ml). Ophthalmologic evaluations included BCVA test, ocular examination, intraocular pressure (IOP) measurement, optical coherence tomography (OCT) imaging and fluorescein angiography (FFA). RESULTS: There were no ocular or systemic adverse events observed, except a mild elevation of IOP [26 mm Hg (1 mm Hg = 0.133 kPa)] in 1 case on the 3rd day after injection, which was controlled by topical medication. One out of 5 eyes had a significant improvement of BCVA (from 0.1 improved to 0.4) in one week after injection. By 2 months, the BCVA increased in 4 cases (increased 1 to 6 lines) and 3 of them remained stable for 4 to 6 months and 1 decreased at the 4 th month post injection. The thickness of central retina reduced 5.9% to 41.4% and FFA revealed a remarkable reduction or an absence of leakage from CNV in 3 eyes by the 4th month. CONCLUSION: Our preliminary results are promising, showing that intravitreous bevacizumab therapy is well tolerated with an improvement in VA, OCT, and FFA outcomes. A multi-center randomized controlled clinical trial is needed to evaluate the long-term safety and effectiveness of intravitreous bevacizumab therapy on neovascular ARMD.

[Clinical study of photodynamic therapy with visudyne for polypoidal choroidal vasculopathy].

OBJECTIVE: To study the clinical effect of photodynamic therapy (PDT) with Visudyne for polypoidal choroidal vasculopathy (PCV). METHODS: Ten patients (10 eyes) with PCV who were diagnosed by fundus fluorescein angiography (FFA), indocyanine green angiography (ICGA), optic coherence tomography (OCT) were treated by PDT with Visudyne. Eight cases (8 eyes) were male, the other two cases (2 eyes) were female. Their ages ranged from 50 to 77 years, mean (59.5 +/- 9.70) years. The best corrected visual activity (BCVA) before PDT was 0.1 - 0.5. The changes of BCVA, fundus photography, FFA and ICGA before and after PDT were compared. Follow-up time varied from 6 months to 36 months, mean 24 months. RESULTS: 1 month after PDT the BCVA was found to be unchanged in 4 eyes, increased in 1 line in 3 eyes, increased in 2 lines in 2 eyes, decreased in 3 lines in 1 eye. FFA and/or ICGA showed no leakage in 4 eyes, leakage reduced in 3 eyes, slight leakage in 2 eyes. At the last follow-up, the BCVA was found to be unchanged in 4 eyes, increased in 1 line in 2 eyes, increased in 2 lines in 2 eyes, increased in 3 lines in 1 eye which received 3 times PDT, decreased in 2 lines in 1 eye. FFA/ICGA showed no leakage in 7 eyes, slight leakage in 2 eyes. No systemic or local adverse effect was found during or after PDT, except 1 eye with extensive subretinal hemorrhage suffered vitreous hemorrhage one month after PDT. CONCLUSIONS: Photodynamic therapy with Visudyne may stop or reduce the macular leakage, facilitate the absorption of hemorrhage, exudates and edema, stabilize or increase the patients' visual activities. It could be a choice for the treatment of PCV. Certainly these tendencies need to be confirmed in a multi-center randomized controlled investigation with longer follow-up time.

Reduced Macular Vascular Density in Myopic Eyes.

BACKGROUND: Morphological changes of the vasculature system in patients with myopia have been observed by Doppler ultrasound and fundus fluorescein angiography (FFA); however, these studies have limitations. Doppler ultrasound provides low-resolution images which are mainly obtained from visualized large vessels, and FFA is an invasive examination. Optic coherence tomography (OCT) angiography is a noninvasive, high-resolution measurement for vascular density. The purpose of this study was to investigate the change of vascular density in myopic eyes using OCT angiography. METHODS: This cross-sectional study includes a total of 91 eyes from 47 participants including control, moderate, and high myopia that were evaluated by OCT angiography. Patients with myopia were recruited from the Refractive Department, Shenzhen Aier Eye Hospital, from August 5, 2015 to April 1, 2016. Emmetropic eyes were from healthy volunteers. The vascular density at macula and optic disc regions, ganglion cell complex (GCC) thickness, and retinal nerve fiber layer (RNFL) thickness were measured. Their relationships with axial length (AL) and refractive error were analyzed. One-way analysis of variance (ANOVA), Pearson's correlation, and generalized estimating equation were used for statistical analysis. RESULTS: Both superficial and deep macular vascular density were highest in control (25.64% ± 3.76% and 37.12% ± 3.66%, respectively), then in moderate myopia (21.15% ± 5.33% and 35.35% ± 5.50%, respectively), and lowest in high myopia group (19.64% ± 3.87% and 32.81% ± 6.29%, respectively) (F = 13.74 and 4.57, respectively; both P < 0.001). Both superficial (ß = -0.850 and 0.460, respectively) and deep (ß = -0.766 and 0.396, respectively) macular vascular density were associated with AL and spherical equivalent (all P < 0.001). Superficial macular vascular density was associated with GCC thickness (ß = 0.244, P = 0.040), independent of spherical equivalent. The vascular density in optic disc region had no difference among the three groups, and it was not associated with AL, spherical equivalent, or RNFL thickness. CONCLUSION: Our results suggested that with the increase of myopia, the vascular density decreased in macular region, but not in optic disc region.

[Gene regulatory mechanism of hairy and enhancer of split related-1 for angiogenesis factors].

OBJECTIVE: To study the roles of hairy and enhancer of split related-1 (HESR-1) in maintenance of the mature, quiescent vessel and angiogenesis. METHODS: Human umbilical vein endothelial cells (HUVEC) were cultured. The full-length coding sequence of HESR-1 was cloned into PcDNA3.1 + using standard protocols. HESR-1 specific siRNA was synthesized and cloned into the RNAi-Ready pSIREN-RetroQ ZsGreen Vector. The constructed PcDNA3.1 + HESR-1 plasmid were transfected into HUVEC for the overexpression of HESR-1, and HESR-1-RNAi plasmid were transfected into HUVEC to silence the HESR-1 gene, the expression of KDR, ALK-1 and Ang-1 in HUVEC were analyzed by RT-PCR and Western blot. RESULTS: The expression of KDR was down-regulated and ALK-1 and Ang-1 were up-regulated in HUVEC with the overexpression of HESR-1; The expression of KDR was up-regulated and ALK-1 and Ang-1 were down-regulated the HESR-1 in HUVEC by RNAi. CONCLUSION: HESR-1 may play an important role in maintenance of vessel in quiescent and control angiogenesis by the regulation of the expression of KDR, ALK-1 and Ang-1.