This study aimed to compare the ameliorative effects of Lycii Fructus and Chrysanthemi Flos at different ratios on retinal damage in mice and to elucidate the underlying mechanisms. A retinal injury model was established by intraperitoneal injection of sodium iodate(NaIO_3) solution. The mice were divided into the following groups: blank group, model group, positive drug(AREDS 2) group, low-and high-dose groups of Lycii Fructus and Chrysanthemi Flos at 1∶1, low-and high-dose groups at 3∶1, and low-and high-dose groups at 1∶3. Administration was carried out 15 days after modeling. The visual acuity of the mice was assessed using the black-and-white box test. The fundus was observed using an optical coherence tomography device, and retinal thickness was measured. HE staining was used to observe the morphology and pathological changes of the retina. The levels of oxidative factors in serum and ocular tissues were measured using assay kits. The levels of inflammatory factors in serum and ocular tissues were detected by enzyme-linked immunosorbent assay(ELISA), and the expression of Nrf2, HO-1, and NF-κB proteins in ocular tissues was analyzed by Western blot. The results showed that after administration of Lycii Fructus and Chrysanthemi Flos at different ratios, the model group showed improved retinal thinning and disordered arrangement of retinal layers, elevated content of SOD and GSH in the serum and ocular tissues, and reduced levels of MDA, TNF-α, IL-1β, and IL-6. Lycii Fructus and Chrysanthemi Flos at 1∶1 and 1∶3 showed better improvement effects. The combination significantly upregulated the expression levels of Nrf2 and HO-1 and downregulated the expression of NF-κB p65. These results indicate that Lycii Fructus and Chrysanthemi Flos at different ratios can improve retinal damage, reduce oxidative stress, and alleviate inflammation in both the body and ocular tissues of mice. The mechanism may be related to the regulation of the Nrf2/HO-1 and NF-κB signaling pathways in ocular tissues. These findings provide a theoretical basis for the clinical application of Lycii Fructus and Chrysanthemi Flos in the treatment of dry age-related macular degeneration.
Animals ; Mice ; Retina/injuries* ; Male ; Lycium/chemistry* ; Drugs, Chinese Herbal/administration & dosage* ; Chrysanthemum/chemistry* ; NF-kappa B/genetics* ; Humans ; Retinal Diseases/metabolism* ; NF-E2-Related Factor 2/metabolism* ; Oxidative Stress/drug effects* ; Flowers/chemistry* ; Heme Oxygenase-1/genetics*

OBJECTIVE: To investigate the the effects of leptin on the proliferation, differentiation and PTEN expression of rat retinal progenitor cells (RPCs) cultured under hypoxic condition. METHODS: SD rat RPCs were cultured in normoxic conditions or exposed to hypoxia in the presence of 0, 0.3, 1.0, 3.0, 10, and 30 nmol/L leptin for 12, 48 and 72 h, and the cell viability was assessed using cell counting kit 8 (CCK 8) assay. The RPCs in primary culture were divided into control group, hypoxia group, and hypoxia+leptin group, and after 48 h of culture, the cell medium was replaced with differentiation medium and the cells were further cultured for 6 days. Immunofluorescence staining was employed to detect the cells positive for β-tubulin III and GFAP, and Western blotting was used to examine the expression of PTEN at 48 h of cell culture. RESULTS: The first generation of RPCs showed suspended growth in the medium with abundant and bright cellular plasma and formed mulberry like cell spheres after 2 days of culture. Treatment with low-dose leptin (below 3.0 nmol/L) for 48 h obviously improved the viability of RPCs cultured in hypoxia, while at high concentrations (above 10 nmol/L), leptin significantly suppressed the cell viability (P < 0.05). The cells treated with 3.0 nmol/L leptin for 48 h showed the highest viability (P < 0.05). After treatment with 3.0 nmol/L leptin for 48 h, the cells with hypoxic exposure showed similar GFAP and β-tubulin Ⅲ positivity with the control cells (P>0.05), but exhibited an obvious down-regulation of PTEN protein expression compared with the control cells (P < 0.05). CONCLUSION In rat RPCs with hypoxic exposure, treatment with low dose leptin can promote the cell proliferation and suppress cellular PTEN protein expression without causing significant effects on cell differentiation.
Animals ; Cell Differentiation/drug effects* ; Cell Hypoxia/drug effects* ; Cell Proliferation/drug effects* ; Cells, Cultured ; Leptin/pharmacology* ; PTEN Phosphohydrolase/metabolism* ; Rats ; Rats, Sprague-Dawley ; Retina/metabolism* ; Stem Cells/metabolism* ; Tubulin

OBJECTIVE: To clarify whether lycium barbarum polysaccharides (LBP) have protective effects on retina neuronal cells in diabetic rats and to identify the related mechanism involved in this process. METHODS: Eighteen SD rats were randomly divided into 3 groups ( n= 6): normal control group (NC), diabetes mellitus group (DM) and LBP-treatment group (DM+LBP). The diabetic rat model was induced by single intraperitoneal injection of streptozotocin (STZ). The rats in DM+LBP group were treated with LBP at the dose of 1 mg/kg by gavage, once a day for 12 weeks. After the treatment, the weight and blood glucose, the generation of reactive oxygen species (ROS), the surviving retinal ganglion cells (RGCs) and amacrine cells and the protein expressions of nuclear factor E2-related factor 2 (Nrf2) and the heme oxygenase-1 (HO-1) were detected. RESULTS: The successful rate of diabetic model was 100%. Compared with NC group, the rats of DM group caused weight loss, elevated blood glucose, a marked increase of ROS generation and a significant decrease in the number of RGCs and amacrine cells (P＜0.01), and these effects were diminished or abolished by LBP treatment. Meanwhile, LBP significantly increased the expressions of Nrf2 and HO-1 in the retinas of diabetic rats (P＜0.01). CONCLUSION LBP can improve retinal oxidative stress and exert beneficial neuroprotective effects in diabetic rats, and its mechanism may be associated with the activation of the Nrf2/HO-1 antioxidant pathway.
Animals ; Diabetes Mellitus, Experimental ; Drugs, Chinese Herbal ; pharmacology ; Heme Oxygenase (Decyclizing) ; drug effects ; NF-E2-Related Factor 2 ; drug effects ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Retina ; drug effects

The aim of this paper was to investigate the preventive effects of Keluoxin Capsules(KLX) on diabetic retinopathy in db/db mice. One hundred male db/db diabetic mice(45-55 g, 8 weeks) were randomly divided into 5 groups(model, KLX low dose, KLX middle dose, KLX high dose, Dobesilate) and 20 male C57 BL/KsJdb~(+/+) were taken as control group. Body weight and fasting blood-glucose were detected every week. Mice were administrated with saline(control and model group), KLX(780, 1 560, 3 120 mg·kg~(-1)·d~(-1), ig), Dobesilate(195 mg·kg~(-1)·d~(-1), ig) for 20 weeks, respectively. At the end of the administration, optical coherence tomography, fundus fluorescein angiography and electroretinogram of the retina were measured. The eyeball was extirpated and retina was isolated to make paraffin section, followed by HE staining and glial fibrillary acidic protein(GFAP) immunohistochemistry. The results indicated that KLX has no obvious effect on body weight and fasting blood level in db/db mice. However, KLX could significantly regulate the thickness of retinal ganglion layer and inner plexiform layer. KLX was able to remarkably reduce the quantity of diabetic microvessel. Meanwhile, KLX could notably improve retinal function. Moreover, KLX could observably modulate the cell arrangement and edema in each layer. There was no markable difference in retina according to the immunochemistry assay. In the present study, KLX exert marked preventive effects on diabetic retinopathy in db/db mice, which provided an experimental evidence for clinical use.
Animals ; Capsules ; Diabetes Mellitus, Experimental ; Diabetic Retinopathy ; drug therapy ; Fluorescein Angiography ; Hypoglycemic Agents ; pharmacology ; Male ; Mice ; Random Allocation ; Retina ; drug effects

The present study was designed to analyze the metabolites of all-trans-retinal (atRal) and compare the cytotoxicity of atRal versus its derivative all-trans-retinoic acid (atRA) in human retinal pigment epithelial (RPE) cells. We confirmed that atRA was produced in normal pig neural retina and RPE. The amount of all-trans-retinol (atROL) converted from atRal was about 2.7 times that of atRal-derived atRA after incubating RPE cells with 10 μmol/L atRal for 24 h, whereas atRA in medium supernatant is more plentiful (91 vs. 29 pmol/mL), suggesting that atRA conversion facilitates elimination of excess atRal in the retina. Moreover, we found that mRNA expression of retinoic acid-specific hydroxylase CYP26b1 was dose-dependently up-regulated by atRal exposure in RPE cells, indicating that atRA inactivation may be also initiated in atRal-accumulated RPE cells. Our data show that atRA-caused viability inhibition was evidently reduced compared with the equal concentration of its precursor atRal. Excess accumulation of atRal provoked intracellular reactive oxygen species (ROS) overproduction, heme oxygenase-1 (HO-1) expression, and increased cleaved poly(ADP-ribose) polymerase 1 (PARP1) expression in RPE cells. In contrast, comparable dosage of atRA-induced oxidative stress was much weaker, and it could not activate apoptosis in RPE cells. These results suggest that atRA generation is an antidotal metabolism pathway for atRal in the retina. Moreover, we found that in the eyes of ABCA4^-/-RDH8^-/- mice, a mouse model with atRal accumulation in the retina, the atRA content was almost the same as that in the wild type. It is possible that atRal accumulation simultaneously and equally promotes atRA synthesis and clearance in eyes of ABCA4^-/-RDH8^-/- mice, thus inhibiting the further increase of atRA in the retina. Our present study provides further insights into atRal clearance in the retina.
ATP-Binding Cassette Transporters/physiology* ; Alcohol Oxidoreductases/physiology* ; Animals ; Cell Survival/drug effects* ; Cells, Cultured ; Humans ; Inactivation, Metabolic ; Mice ; Retina/metabolism* ; Retinal Pigment Epithelium/metabolism* ; Swine ; Tretinoin/pharmacology*

Background: The demonstrated role of mitogen-activated protein kinase (MAPK) in both cell apoptosis and the inflammation pathway makes it an attractive target for photoreceptor protection. The aim of this study was to investigate the protective effects of MAPK antagonists against photoreceptor degeneration and retinal inflammation in a rat model of light-induced retinal degeneration. Methods: Sprague Dawley rats were treated with intravitreal injections of MAPK antagonists, inhibitors of p-P38, phosphorylated-extracellular regulated kinase (p-ERK) 1/2, and p-c-Jun N-terminal kinase (JNK) just before they were assigned to dark adaptation. After dark adaptation for 24 h, rats were exposed to blue light (2500 lux) in a light box for 24 h, and then returned to the normal 12-h light/12-h dark cycle. Samples were collected at different time points. MAPK expression during light exposure was examined with immunofluorescence. Photoreceptor death was detected with histopathology and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The expression of retinal p-ERK1/2, caspase 3, activated caspase 3, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β was examined by Western blotting. Differences between groups were evaluated using unpaired one-way analysis of variance and least significant difference post hoc tests. Results: MAPKs (P38, ERK1/2, and p-JNK) were phosphorylated and activated in the light injury groups, compared with normal group, and their expressions were mainly elevated in the outer nuclear layer (ONL). Among the selected MAPK antagonists, only the p-ERK1/2 inhibitor attenuated the loss of photoreceptors and the thinning of ONL in light injury groups. Besides, p-ERK1/2 inhibitor refrained light-induced photoreceptor apoptosis, which was presented by TUNEL positive cells. Light injury significantly increased the expression of p-ERK1/2 (1.12 ± 0.06 vs. 0.57 ± 0.08, t = 9.99, P < 0.05; 1.23 ± 0.03 vs. 0.57 ± 0.08, t = 11.90, P < 0.05; and 1.12 ± 0.12 vs. 0.57 ± 0.08, t = 9.86, P < 0.05; F = 49.55, P < 0.001), and induced caspase 3 activating (0.63 ± 0.06 vs. 0.14 ± 0.05, t = 13.67, P < 0.05; 0.74 ± 0.05 vs. 0.14 ± 0.05, t = 16.87, P < 0.05; and 0.80 ± 0.05 vs. 0.14 ± 0.05, t = 18.57, P < 0.05; F = 100.15, P < 0.001), compared with normal group. The p-ERK1/2 inhibitor significantly reduced p-ERK1/2 overexpression (0.61 ± 0.06 vs. 1.12 ± 0.06, t = -9.26, P < 0.05; 0.77 ± 0.06 vs. 1.23 ± 0.03, t = -8.29, P < 0.05; and 0.68 ± 0.03 vs. 1.12 ± 0.12, t = -7.83, P < 0.05; F = 49.55, P < 0.001) and downregulated caspase 3 activating (0.23 ± 0.04 vs. 0.63 ± 0.06, t = -11.24, P < 0.05; 0.43 ± 0.03 vs. 0.74 ± 0.05, t = -8.86, P < 0.05; and 0.58 ± 0.03 vs. 0.80 ± 0.05, t = -6.17, P < 0.05; F = 100.15, P < 0.001), compared with light injury group. No significant change in the total level of caspase 3 was seen in different groups (F = 0.56, P = 0.75). As for inflammation, light injury significantly increased the expression of TNF-α (0.42 ± 0.04 vs. 0.25 ± 0.05, t = 5.99, P < 0.05; 0.65 ± 0.03 vs. 0.25 ± 0.05, t = 14.87, P < 0.05; and 0.86 ± 0.04 vs. 0.25 ± 0.05, t = 22.58, P < 0.05; F = 160.27, P < 0.001) and IL-1β (0.24 ± 0.01 vs. 0.19 ± 0.02, t = 2.33, P < 0.05; 0.35 ± 0.02 vs. 0.19 ± 0.02, t = 7.97, P < 0.05; and 0.48 ± 0.04 vs. 0.19 ± 0.02, t = 14.69, P < 0.05; F = 77.29, P < 0.001), compared with normal group. P-ERK1/2 inhibitor significantly decreased the overexpression of TNF-α (0.22 ± 0.02 vs. 0.42 ± 0.04, t = -7.40, P < 0.05; 0.27 ± 0.02 vs. 0.65 ± 0.03, t = -14.27, P < 0.05; and 0.33 ± 0.03 vs. 0.86 ± 0.04, t = -19.58, P < 0.05; F = 160.27, P < 0.001) and IL-1β (0.13 ± 0.03 vs. 0.24 ± 0.01, t = -5.77, P < 0.05; 0.17 ± 0.01 vs. 0.22 ± 0.02, t = -9.18, P < 0.05; and 0.76 ± 0.05 vs. 0.48 ± 0.04, t = -13.12, P < 0.05; F = 77.29, P < 0.001), compared with light injury group. Conclusion The p-ERK1/2 inhibitor might protect the retina from light-induced photoreceptor degeneration and retinal inflammation.
Animals ; Blotting, Western ; In Situ Nick-End Labeling ; Interleukin-1beta ; metabolism ; Light ; Male ; Mitogen-Activated Protein Kinases ; metabolism ; Phosphorylation ; drug effects ; Rats ; Rats, Sprague-Dawley ; Retina ; drug effects ; metabolism ; Retinal Degeneration ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate whether propranolol application as collyrium or intraperitoneal (IP) injection can promote the recovery of oxygen-induced retinopathy (OIR).

METHODThirty-six 7-day-old mice were divided into the following 6 groups: normal control, propranolol eye drops, propranolol IP injection, eye drops negative control, IP injection negative control, and pathological model with 6 mice in each. In a typical model of OIR, litters of mice pups with their nursing mothers were exposed to an infant incubator to high oxygen concentration (75 ± 5)% between postnatal day (PD) 7 and PD12, prior to returning to room air. Two routes of propranolol treatment were assessed from PD12 to PD17: IP injection and eye drop, with doses 2 mg/(kg·time), three times a day. Another three groups were given citric acid buffer eye drops, IP injection of citric acid buffer, and negative control were not treated with any drug. Neonatal mice fed in normal conditions served as normal control. Mice were sacrificed at PD17 to evaluate the morphological changes of retinal vessels by fluorescein isothiocyanate-dextran perfusion and retinal whole mount. The retinal neovascularization was evaluated by counting the number of nuclei of the endothelial cell breaking through the internal limiting membrane (ILM).

RESULTCompared with the oxygen-exposed group, the branches of retinal vessels went normal with a less un-perfused area in the propranolol eye drops and propranolol IP injection groups [(38.9 ± 9.9)% and (5.6 ± 2.3)% vs. (16.2 ± 10.0)% and (2.2 ± 0.8)%, (25.9 ± 5.0)% and (2.1 ± 2.7)%, F=36.12 and 14.55, P both<0.001]. The number of nuclei of endothelial cells breaking through the ILM on the retinal cross-section in the propranolol eye drops group decreased (14.2 ± 5.1) per slide, which was less than that in the oxygen-exposed group (49.1 ± 8.9) per slide and the propranolol IP injection group (18.0 ± 5.9) per slide; it was also less than that in the eye drops negative control group (47.4 ± 8.1) per slide (F=187.60, P<0.05). Moreover, the number of nuclei of endothelial cells breaking through the ILM on the retinal cross-section in the propranolol IP injection group was less than that in the IP injection negative control group (49.9 ± 7.1) per slide (P<0.05).

CONCLUSIONPropranolol could effectively inhibit the formation of retinal neovascularization in mice; the eye drops was more effective than the IP injection.

Animals ; Dextrans ; Disease Models, Animal ; Endothelial Cells ; Fluorescein-5-isothiocyanate ; analogs & derivatives ; Injections, Intraperitoneal ; Mice ; Ophthalmic Solutions ; Oxygen ; adverse effects ; Propranolol ; therapeutic use ; Retina ; drug effects ; Retinal Neovascularization ; chemically induced ; drug therapy ; prevention & control ; Retinal Vessels ; drug effects

BACKGROUNDRetinal degenerative diseases are the leading causes of blindness in developed world. Retinal progenitor cells (RPCs) play a key role in retina restoration. Triamcinolone acetonide (TA) is widely used for the treatment of retinal degenerative diseases. In this study, we investigated the role of TA on RPCs in hypoxia condition.

METHODSRPCs were primary cultured and identified by immunofluorescence staining. Cells were cultured under normoxia, hypoxia 6 h, and hypoxia 6 h with TA treatment conditions. For the TA treatment groups, after being cultured under hypoxia condition for 6 h, RPCs were treated with different concentrations of TA for 48-72 h. Cell viability was measured by cell counting kit-8 (CCK-8) assay. Cell cycle was detected by flow cytometry. Western blotting was employed to examine the expression of cyclin D1, Akt, p-Akt, nuclear factor (NF)-κB p65, and caspase-3.

RESULTSCCK-8 assays indicated that the viability of RPCs treated with 0.01 mg/ml TA in hypoxia group was improved after 48 h, comparing with control group (P < 0.05). After 72 h, the cell viability was enhanced in both 0.01 mg/ml and 0.02 mg/ml TA groups compared with control group (all P < 0.05). Flow cytometry revealed that there were more cells in S-phase in hypoxia 6 h group than in normoxia control group (P < 0.05). RPCs in S and G2/M phases decreased in groups given TA, comparing with other groups (all P < 0.05). There was no significant difference in the total Akt protein expression among different groups, whereas upregulation of p-Akt and NF-κB p65 protein expression and downregulation of caspase-3 and cyclin D1 protein expression were observed in 0.01 mg/ml TA group, comparing with hypoxia 6 h group and control group (all P < 0.05).

CONCLUSIONLow-dose TA has anti-apoptosis effect on RPCs while it has no stimulatory effect on cell proliferation.

Animals ; Apoptosis ; drug effects ; physiology ; Caspase 3 ; metabolism ; Cell Cycle ; drug effects ; physiology ; Cell Hypoxia ; drug effects ; physiology ; Cell Proliferation ; drug effects ; physiology ; Cell Survival ; drug effects ; physiology ; Cells, Cultured ; Cyclin D1 ; metabolism ; NF-kappa B ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Rats, Sprague-Dawley ; Retina ; cytology ; Stem Cells ; cytology ; drug effects ; Triamcinolone Acetonide ; pharmacology

PURPOSE: To study the retinal pigment epithelium (RPE) and retinal alterations in chronic central serous chorioretinopathy treated with photodynamic therapy, and its correlation with functional parameters such as best-corrected visual acuity (BCVA) and contrast sensitivity (CS). METHODS: Retrospective, noncomparative, consecutive evaluation by optical coherence tomography and its correlation with BCVA and CS in 31 eyes of 26 patients. RESULTS: In all affected patients, 88.5% were male with a mean age of 42.9 years. The right eye was involved in 64.5% of cases, bilateral in 19% and 73.9% were hyperopic (spherical refraction between 0 and +5.0 diopters). Of these cases, 51.5% had peri-RPE abnormalities, 17.3% hyperreflective substances at RPE, 19.4% RPE atrophy, 55.3% foveolar atrophy, 3.1% pigment epithelial detachment, 5.2% subretinal fluid persistence, 8.3% fibrin deposits, 68.4% photoreceptor inner and outer segment line interruption and 31.1% external limiting membrane interruption. CONCLUSIONS: Time evolution and number of outbreaks were related to the decrease in foveal and chorodial thickness and in those with worse BCVA and CS. RPE abnormalities and atrophy were related to the age of onset of symptoms. Photoreceptor elongation has been correlated with poor BCVA and inner and outer segment line destructuring and interruption with poor CS.
Adult ; Central Serous Chorioretinopathy/diagnosis/*drug therapy/physiopathology ; Chronic Disease ; Female ; Fluorescein Angiography ; Follow-Up Studies ; Fundus Oculi ; Humans ; Male ; Middle Aged ; Photochemotherapy/*methods ; Photosensitizing Agents/administration & dosage ; Porphyrins/*administration & dosage ; Retina/*diagnostic imaging/drug effects/physiopathology ; Retrospective Studies ; Tomography, Optical Coherence ; Treatment Outcome ; *Visual Acuity

PURPOSE: To investigate the effects of vitreomacular traction (VMT) on ranibizumab treatment response for neovascular age-related macular degeneration (AMD). METHODS: A retrospective review of 85 eyes of 85 patients newly diagnosed with neovascular AMD was conducted. Patients were eligible if they had received more than three consecutive monthly ranibizumab (0.50 mg) treatments and ophthalmic evaluations. Patients were classified into a VMT (+) group or VMT (-) group according to optical coherence tomography imaging. Best corrected visual acuity and central retinal thickness (CRT) measurements were obtained at three and six months after initial injection. RESULTS: One month after the third injection, mean visual acuity (VA) increases of 6.36 and 9.87 letters were observed in the VMT (+) and VMT (-) groups, respectively. The corresponding mean CRT values decreased by 70.29 microm and 121.68 microm, respectively. A total 41 eyes were identified as eligible for a subsequent fourth injection; 71.1% of patients (27 eyes) in the VMT (+) group but only 29.8% of patients in the VMT (-) group needed a subsequent fourth injection. Follow-up was extended to six months for 42 of the 85 enrolled patients (49.4%). The trends in VA and optical coherence tomography were found to be maintained at six-month follow-up. CONCLUSIONS: VA and CRT appeared to be more improved after ranibizumab treatment in the VMT (-) group compared to the VMT (+) group. VMT might antagonize the effect of ranibizumab treatment in a subpopulation of AMD patients.
Aged ; Aged, 80 and over ; Angiogenesis Inhibitors/*therapeutic use ; Female ; Follow-Up Studies ; Humans ; Intravitreal Injections ; Male ; Middle Aged ; Ranibizumab/*therapeutic use ; Retina/pathology ; Retinal Diseases/*physiopathology ; Retrospective Studies ; Tissue Adhesions ; Tomography, Optical Coherence ; Vascular Endothelial Growth Factor A/antagonists & inhibitors ; Visual Acuity/drug effects ; Vitreous Body/*pathology ; Wet Macular Degeneration/*drug therapy/physiopathology