Objective To investigate the protective effect of intravitreal injection of nerve growth factor (NGF) on apoptosis of retinal ganglion cells (RGCs) in early diabetic rats and its possible mechanism.Methods SD rats were divided into normal control group,diabetic model group,phosphate buffer solution (PBS) group and NGF group according to random number table method,with each group contain 6 rats.The rats in diabetic model group,PBS group and NGF group were injected intraperitoneally with streptozotocin (STZ) to establish diabetic rat model.After 4 weeks of modeling,2 μl PBS and 2 μl NGF (0.5 μg/μl) were injected into the vitreous cavity in PBS group and NGF group,respectively.The right eyes served as the experimental eyes,inject once a week for 4 weeks.After 4 weeks of injection,the retina microangiopathy of each group was observed by Phoenix Micron Ⅳ small animal retinal imaging system.After high dose anesthesia,the eyeball of one rat from each group was taken to prepare ultrathin sections and observed by transmission electron microscopy.Five rat eyeballs were taken to prepare retinal paraffin sections from each groups.The RGC apoptotic index was observed by TUNEL method.The expressions of RGC bcl-2 protein and bax protein were analyzed by immunohistochemistry.This study was approved by the Experimental Animal Ethics Committee of Central South University,and the experimental procedures were in accordance with the National Institutes of Health(NIH) guidelines for the Care and Use of Laboratory,and follow the 3R principle.Results The weight of the diabetic model rats was significantly lower than that of the normal group,and the intake of water and food were significantly increased,the urine volume was also in creased.The body weight and blood glucose level of the rats were significantly different among different groups after 4 weeks of modeling (F=202.352,148.444,both at P＜0.001).Fundus photography of early diabetic rats showed no obvious diabetic retinal microangiopathy.The numbers of RGC in the diabetic model group and PBS group were significantly lower than that in the normal control group under the transmission electron microscope.The membrane shrinkage,cytoplasmic condensation,organelle edema,chromatin peripheral collection were obvious and the cell apoptosis number was increased.The RGC lesions in the NGF group were lighter than those in the diabetic model group and PBS group.The apoptotic indexs in the normal control group,diabetic model group,PBS group and NGF group were (3.88±1.28)％,(92.56±1.58)％,(92.64±2.30)％ and (59.34±3.89)％,respectively,the overall difference of apoptotic index between the four groups was statistically significant (F=854.554,P＜0.001);the RGC apoptotic index of the diabetic model group was significantly higher than that of the normal control group.The RGC apoptotic index of the NGF group was significantly lower than that of the diabetic model group and PBS group (both at P＜0.05).The expression levels of bcl-2 protein in normal control group,diabetic model group,PBS group and NGF group were 38.11 ± 1.01,22.38±3.90,23.04±3.14 and 84.69±1.45,respectively,with a significant difference among the groups (F =366.206,P＜0.001).The expression levels of bax protein in normal control group,diabetes model group,PBS group and NGF group were 4.22± 1.89,56.59±6.67,56.30±8.51 and 26.19±2.44,respectively,with a significant difference among the groups (F=61.435,P＜0.001).The expression of bcl-2 protein in the diabetic model group was significantly lower than that in the normal control group (P＜0.05),and the expression of bax protein was significantly increased (P＜0.05).The expression of bcl-2 protein in NGF group was significantly higher and the expression of bax protein was significantly lower than those in the PBS group and diabetic model group (all at P＜0.05).Conclusions The ultrastructural damage of RGCs has occurred in early diabetic rats without diabetic retinal microangiopathy.Intravitreal injection of NGF may produce retinal neuroprotective effects by inhibiting apoptosis of RGCs in diabetic rats.

The occurrence of high intraocular pressure (IOP) after vitrectomy for diabetic retinopathy (DR) is related to many factors,including the type and stage of DR,macular detachment,surgical methods,and the type of ocular tamponade.Early high IOP occurred mainly due to laser photocoagulation,inflammatory response,improper ocular tamponade,residual viscoelastic agents and ciliary body dysfunction.In addition to the above reasons,early-middle stage high IOP is also related to tamponade gas expansion peak,encircling scleral buckle and hyphema.The major reason for middle-stage high IOP is hyphema and silicon oil in anterior chamber.The reasons for late-stage high IOP are glaucoma,silicone oil emulsification,long-term use of glucocorticoid,and iris incision closure.Most high IOP can be controlled by proper treatment such as stopping use of glucocorticoid,anti-glaucoma eye drops and surgeries.But there are still a small number of patients with unexplained refractory high IOP,the mechanism need to be further explored.

OBJECTIVE: To compare the difference in intraocular pressure (IOP) readings as well as the tolerability between Icare rebound tonometer (Icare RBT) and Goldmann applanation tonometer (GAT), and to evaluate the application of Icare RBT in monitoring the intraocular pressure in children after congenital cataract surgery. METHODS: The IOP was measured with the Icare RBT and GAT respectively in 150 children (262 eyes) after congenital cataract surgery by two experienced ophthalmologists. Correlation and Bland-Altman analysis were used to assess the agreement in IOP readings between the two instruments. The influence of the central corneal thickness (CCT) adjusted for age on IOP readings was analyzed by linear regression analysis. The tolerance of the patients to Icare RBT and GAT measurement were surveyed. RESULTS: The mean age was (44.82 ± 11.56) months in 150 children, including 81 boys and 69 girls. The mean IOP readings by the Icare RBT and GAT were (16.08 ± 5.72) mmHg and (14.17 ± 5.05) mmHg, respectively. The mean difference between the Icare RBT and GAT was (1.91 ± 2.04) mmHg, which was significantly correlated with CCT (r=0.409, P<0.001). The IOP readings by Icare RBT was significantly correlated with that measured by GAT(r= 0.936, P<0.001). The 95% confidence interval of the difference between the two instruments was ?2.10 to 5.91 mmHg. The Icare RBT examination was well tolerated by the children compared to the GAT examination. CONCLUSION The Icare RBT is easy to use and well tolerated by the children after congenital cataract surgery. Compared to GAT, the value measured by the IOPs trends to be overestimated. The difference in readings between the 2 tonometers will magnify with the increase in CCT.
Cataract ; congenital ; Cataract Extraction ; Child, Preschool ; Female ; Humans ; Intraocular Pressure ; Male ; Tonometry, Ocular ; instrumentation

Background One of the important machanisms of all trans retinoic acid (ATRA) is to regulate the expression of connexin (Cx) gene.ATRA inhibits the proliferation and differentiation of retinoblastoma (RB) cells,which is related to Cx43.However,the control site of ATRA and its effect on RB tumor in vivo have not been identified.Objective This study was to investigate the effect of ATRA on Cx43 expression in RB cells and its approach mechanisms.Methods ATRA solution of 1 × 10 2 mol/L was prepared with ethanol and formulated into 1×10 5,1×10-6and 1 × 10 7 mol/L of solution with culture medium further.Human RB cell line (HXO-RB44) was cultured and treated with different concentrations of ATRA for 2,4 and 6 days,respectively.The expressions of Cx43 protein and mRNA in RB cells were detected by Western blot and reverse transcription PCR (RT-PCR),respectively.RB models were established by injecting HXO-RB44 cell suspension into anterior chamber in the right eyes of 15 athymic mice.Eleven successful models were divided into the blank control group,negative control group and 1 × 10-5 mol/L ATRA group,and 0.5％ normal saline solution with athymic or 1 ×10-5 mol/L ATRA solution was injected into the anterior chamber in the negative control group and 1 × 10-5 mol/L ATRA group in the 3-day interval for 3 weeks.The model eyes were examined under the slit lamp microscope.The eyeballs were extracted at the end of the experiment for hematoxylin and eosin staining.Results Western blot assay showed that the absorbance values of Cx43 protein (ACx43/AGAPDH) were increased gradually as time lapse of ATRA treatment among the groups (Ftime =71.31,P =0.00; Fgroup =7.66,P =0.00).The expressions of Cx43 protein were significantly higher in the 1 × 10 5 mol/L ATRA group after 2 days,1 × 10-6 mol/L ATRA group after 4 days,1 × 10-7 mol/L ATRA group after 6 days than those in the blank control group at various time points (t =3.34,P＜0.01 ;t =2.33,P＜0.05;t =3.12,P＜ 0.01).RT-PCR showed that the absorbance values of Cx43 mRNA (ACx43mRXA/Aβ-actin) were significantly enhanced as the prolong of treatment time of ATRT among the groups (Ftime =90.90,P =0.00 ; Fgroup =6.86,P =0.00).The expressions of Cx43 mRNA were significantly higher in the 1 × 10-5 mol/L ATRA group after 2 days,1 × 10 6 mol/L ATRA group and 1 ×10-7 mol/L ATRA group after 4 days than those in the blank control group at various time points (t=3.57,P＜0.01 ;t=6.31,P＜0.01 ;t=2.22,P＜0.05).RB models were successfully created in 11 eyes on the 6-9 days following the intrachamber injection of RB cell suspension.The RB cells were filled with chamber in the blank control group 20 days after injection,and RB only occupied half of the anterior chamber in the 1 × 10-5 mol/L ATRA group.Histopathological examination exhibited that the RB cells were seen in the anterior and posterior chamber as well as vitreous in the blank control group,however,the cells were only found in the anterior chamber in the 1 × 10 5 mol/L ATRA group.Conclusions ATRA can inhibit the growth of RB in vitro and in vivo by inducing the expression of Cx43 gene in transcription process.

OBJECTIVE: To determine the apoptosis-inducing effect of ultraviolet light (UV) on human lens epithelial cell (HLEC) and to explore the involvement of changes in ALDH1 folowing UV radiation. METHODS: HLEC was exposed to the same UV light source and was subsequently divided into 6 groups according to UV radiation time of 0 (control group), 5, 10, 15, and 30 min. Apoptosis was detected by AO/EB staining. Changes of ALDH1 in HLEC were detected by immunohistochemical staining and Western blot. RESULTS: The intensity of immunohistochemical staining and the rate of positive cells decreased with increase of UV time (P<0.05). The rate of positive ALDH1 cells was negatively correlated with the rate of apoptosis (r= -0.92, P<0.05). Western blot showed the integrated absorbance values significantly decreased with the increase of UV time (P<0.05). CONCLUSION ALDH1 in HLEC decreases with an increase of UV exposure, which may be related to UV induced apoptosis of HLEC.
Aldehyde Dehydrogenase 1 ; Apoptosis ; radiation effects ; Cells, Cultured ; Epithelial Cells ; cytology ; metabolism ; radiation effects ; Humans ; Isoenzymes ; genetics ; metabolism ; Lens, Crystalline ; cytology ; Retinal Dehydrogenase ; genetics ; metabolism ; Ultraviolet Rays ; adverse effects

OBJECTIVE: To investigate and compare the growth of human fetal retinal pigment epithelial (RPE) cells seeded onto electrospun polyamide nanofibers (EPN) or etched pore polyester (EPP), and, further, to explore their possible use as prosthetic Bruch's membrane. METHODS: Human fetal RPE cells were planted onto the EPN, EPP and plastic (control) substrates in Transwells. The cultures were assessed with respect to cell attachment at 2, 4, 8 hours and proliferation at 1, 4, 8 days after seeding. Growth and morphology of the cells were monitored under the phase contrast microscope, and the phenotype was identified by immunofluorescence staining with antibodies against tight junction protein ZO-1. Strips of single EPP coated with nothing or EPP coated with EPN was differently implanted into the subretinal space of two P21 RCS rats for two weeks and the histologic slides of the retina were assessed. RESULTS: Cultured human fetal RPE cells were attached to either EPN or EPP substrates (with seeding on plastic substrate as control). After 8 h, the numbers of adherent cells in the EPN, EPP and control groups were 1.23*10(5)/cm(2), 1.70*10(5)/cm(2), and 1.64*10(5)/cm(2), respectively. The number of RPE cells attached to EPN was obviously less than that to both EPP and control (P<0.05). On the first day, the proliferation of cells on EPN was less than that of EPP and control (P<0.05); but by the 8th day in culture, the proliferation of cells on EPN had increased and was higher than proliferation on both EPP and control (P<0.05). All of the RPE cells cultured on EPN and EPP substrates were in monolayer, and the EPN-attached cells resembled the inner collagenous layer of Bruch's membrane. Immunofluorescence staining showed that the RPE cells cultured on EPN and EPP substrates adopted a higher expression of ZO-1 than that on the plastic control substrate. Subretinal implantation of either EPP alone or EPP as a carrier for free EPN for 2 weeks in P21RCS rats resulted in an expected encapsulation and loss of photoreceptor layer. No toxicity or other adverse reaction was observed in the vicinity of the transplant. CONCLUSION EPN and EPP could maintain human fetal RPE cell attachment and proliferation. Both EPN and EPP appeared to be grossly tolerance and biocompatible with subretinal implantation. EPN represents an intriguing prospect for prosthetic Bruch's membrane replacement because of its similarity in structure to native Bruch's membrane.
Animals ; Biocompatible Materials ; chemistry ; Bruch Membrane ; Cell Proliferation ; Cells, Cultured ; Fetus ; Humans ; Membranes, Artificial ; Nanofibers ; chemistry ; Polyesters ; chemistry ; Porosity ; Rats ; Retinal Pigment Epithelium ; cytology ; growth & development ; Tissue Engineering

OBJECTIVE: To explore the apoptosis-inducing effect of ultraviolet(UV) radiation on human lens epithelial cells (HLEC), with particular focus on changes in Bcl-2 or Bax expression as possible mechanisms. METHODS: All experimental groups were exposed to the same UV light source. HLEC were divided into 6 groups according to duration of UV radiation : 0 min group (control group), 5 min group, 10 min group,15 min group, and 30 min group. Analysis on apoptosis of HLEC was performed by flow cytometry analysis (FCA, Annexin V + PI staining). Changes of Bax and Bcl-2 expression in HLEC were detected by hybridization in situ. RESULTS: Apoptosis in HLEC increased with UV exposure time. The expression level of Bax mRNA was increased with the increase of UV exposure time, whereas the expression level of Bcl-2 mRNA decreased with the increase of UV exposure time. The proportion of apoptotic cells was negatively correlated with ratio of Bcl-2/Bax (r=-0.874, P<0.05). CONCLUSION UA radiation can induce apoptosis of HLEC in vitro. Bcl-2 and Bax genes may play an important role in regulating this apoptotic process.
Apoptosis ; radiation effects ; Cells, Cultured ; Epithelial Cells ; cytology ; metabolism ; radiation effects ; Humans ; Lens, Crystalline ; cytology ; radiation effects ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Time Factors ; Ultraviolet Rays ; adverse effects ; bcl-2-Associated X Protein ; genetics ; metabolism

We described clinical process of two cases of intraocular lymphoma in aspects of early diagnosis by fine needle aspiration (FNA) and biopsy and treatment by intravitreal methotrexate (MTX). Two patients were suspected to have primary intraocular lymphoma (PIOL) with geographic yellow-white infiltrates and vitreous opacity. FNA confirmed malignant intraocular lymphoma in one patient and failed in the other patient due to complication of vitreous hemorrhage. Subsequent vitreous biopsy confirmed malignant intraocular lymphoma in the other patient. Both patients were treated by intravitreal methotrexate. In case 1 the tumor had complete remission and follow-up of 12 months had not found any signs of recurrence. In case 2 the patient died of brain metastasis 22 months after the ocular biopsy. Our findings demonstrate that although cytological examination of vitrectomy specimens remains the gold standard in diagnosis of PIOL, examination of FNA and biopsy increases the reliability of early diagnosing or excluding a PIOL. Individualized intravitreal methotrexate can be used to effectively treat PIOL. More effective integrated program treating primary central nervous system lymphoma/PIOL is worthy of looking forward to.

We described clinical process of two cases of intraocular lymphoma in aspects of early diagnosis by fime needle aspiration (FNA) and biopsy and treatment by intravitreal methotrexate (MTX).Two patients were suspected to have primary intraocular lymphoma (PIOL) with geographic yellow-white infiltrates and vitreous opacity.FNA confirmed malignant intraocular lymphoma in one patient and failed in the other patient due to complication of vitreous hemorrhage.Subsequent vitreous biopsy confirmed malignant intraocular lymphoma in the other patient.Both patients were treated by intravitreal methotrexate.In case 1 the tumor had complete remission and follow-up of 12 months had not found any signs of recurrence.In case 2 the patient died of brain metastasis 22 months after the ocular biopsy.Our findings demonstrate that although cytological examination of vitrectomy specimens remains the gold standard in diagnosis of PIOL,examination of FNA and biopsy increases the reliability of early diagnosing or excluding a PIOL.Individualized intravitreal methotrexate can be used to effectively treat PIOL.More effective integrated program treating primary central nervous system lymphoma/PIOL is worthy of looking forward to.

OBJECTIVE: To investigate the role of Notch signaling in differentiation of Sprague-Dawley (SD) rat retinal progenitor cells (RPCs). METHODS: RPCs were isolated from 16-day embryonic SD rats and cultured in suspension. RPCs were cultured respectively in media with (treatment group) or without (control group) gamma-secretase inhibitor X which was used to block Notch signaling. Morphological observation and immunocytochemistry were applied at day 14 to determine the cell types and analyze the expression of Notch pathway genes in both groups. RESULTS: Most RPCs expressed Notch1 intracellular domains or its downstream transcriptional factor Hes1. A few expressed bHLH transcriptional factors NeuroD and Mash1. Most auto-differentiated RPCs expressed NeuroD or Mash1, while a few of them expressed Notch1 intracellular domains or Hes1. In the group treated with gamma-secretase inhibitor X, the positive rate of Nestin or GFAP was much lower than that in the control group while the positive rate of beta-tubulin was much higher than that in the control group. The difference in the positive rate of recovering between the two groups was not significant. CONCLUSION In vitro Notch signaling may inhibit retinal stem cells differentiation. Inhibiting Notch signaling in vitro may promote differentiation to neurons and partially inhibit glial differentiation.
Animals ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Cell Differentiation ; physiology ; Cells, Cultured ; Female ; Fetus ; Homeodomain Proteins ; metabolism ; Neurons ; cytology ; Rats ; Rats, Sprague-Dawley ; Receptor, Notch1 ; genetics ; metabolism ; Retina ; cytology ; Signal Transduction ; drug effects ; physiology ; Stem Cells ; cytology ; Transcription Factor HES-1