Objective To observe the effects of basic fibroblast growth factor (bFGF) on the expression of heat shock protein 70 (HSP70) in rat′s retina after ischemia/reperfusion injury. Methods The rat model of experimental retinal ischemia/ reperfusion injury was made by increasing the intraocular pressure. Tweenty-four Wistar rats were divided into normal (3 rats) and operation group (21 rats) randomly. The latter group was subdivided into group 0 hour, 4, 8, 12, 24, 48 and 72 hours after reperfusion, in which the left eyes of the rats were in the ischemia/reperfusion groups and the right ones were in the treatment groups (bFGF 2 ?g intracameral injection). The expression of HSP70 was observed by strept avidin-biotin complex (SABC) immunohistochemistry. Results No HSP70 positive cells were found in normal group; a few of HSP70 positive cells were found 0 hour after reperfusion [20.8?4.5)cells/mm 2], and increased gradually until reached the peak 24 hours later [(111.2?4.4) cells/mm 2] and then decreased gradually. Few HSP70 positive cells were found 72 hours after reperfusion. The amount of HSP70 positive cells increased in treatment group at all time courses, and the peak time was earlier and longer than that in ischemia group. HSP70 positive cells distributed extensively in retinal ganglion cell layer and inner nucleous layer. The difference of the amount of HSP70 positive cells between the two groups was significant ( P

BACKGROUND: Recent studies demonstrate that erythropoietin (EPO) can protect retina from light injury, and is the mechanism related to the expression of Caspase-3 in the light-injured retinal pigment epithelium (RPE) cells. OBJECTIVE: To study the effects of EPO at different dosages on the expression of Caspase-3 in light-injured human RPE cells. DESIGN: Control observation.SETTING: Qingdao University Medical College. MATERIALS: Adult ARPE-19 cells (American Cell Culture Collection Company); DMEM/F12 mixed medium, fetal bovine serum and trypase (GIBCO Biotechnique Company); recombinant human EPO (rhEPO, Sigma Biotechnique Company); human Caspase-3 quantitative kit (Shanghai Xitang Biotechnique Co.,Ltd); Caspase-3 monoclonal antibody (American Santa Cruz Company); PV6001 immunohistochemistry kit and DAB color reagent kit (Zhongshan Biotechnology Company, Beijing).METHODS: The experiment was carried out in the Department of Pathophysiology at Qingdao University Medical College between May 2006 and January 2007. Human RPE-19 cell strain at passages 2-5 were harvested for light injury models, and the passage cells were divided into 7 groups randomly, with 4 apertures in each group:①normal control group: no light or EPO intervention;②light-injured model group: 12-hour illumination, no EPO intervention;③light-injury and EPO groups: 12-hour illumination with 10 000, 20 000 and 40 000 U/L EPO;④light-injury and 40 000 U/L EPO and AG490 group: 12-hour illumination with 40 000 U/L EPO and inhibitor of Jak2 enzyme 50 000 U/L;⑤light-injury and 40 000 U/L EPO and carbxyl-terminal modulator protein (CTMP) group: 12-hour illumination with 40 000 U/L EPO and specific inhibitor of protein kinase B enzyme 100 μmol/L.MAIN OUTCOME MEASURES: The enzyme linked immunosorbant assay (ELISA) and immunohistochemistry were used to assess the effects of rhEPO at the different doses on the expression of Caspase-3 in light-injured human RPE cells. RESULTS: Caspases-3 was not expressed in RPE cells of the normal control group and was positively expressed in the nucleus of RPE cells of the light-injured model group, showing a specific brown-yellow staining. Expression of Caspase-3 was gradually decreased in every rhEPO group with increase of EPO concentration, with the weakest expression in 40 000 U/L rhEPO group. The effects of EPO on Caspase-3 expression were strongly inhibited in light-injury+ 40 000 U/L EPO +AG490 group and the expression was positive in light-injury +40 000 U/L EPO+CTMP group, which was slightly weaker than light-injured model group. CONCLUSION: The rhEPO can reduce the expression of Caspase-3 in the light-injured human RPE cells, and one of the possible mechanisms is the inhibition of light-injured RPE cell apoptosis by the rhEPO.

Objctive To explore the relationship between the expression of Fas/FasL and the apoptosis occurs in retinal ischemia/reperfusion injury of rats, as well as the therapeutic effects of bFGF on the ischemic retina. Methods The models of retinal ischemia/reperfusion injury was made by transient elevating introcular pressure. A total of 28 rats were divided into normal and operation group.The latter were subdivided into 1 hour, 6, 12, 24, 48 and 72 hours after reperfusion group, in which the left eyes of the rats were in the ischemia/reperfusion groups and the right ones were in the treatment groups (bFGF intracameral injection). Apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling (TUNEL) method, and the expression of Fas and Fas ligand was studied by strept avidin-biotin complex (SABC)immunohistochemistry. Results No positive cells were observed in the normal rats′ retinae, but there was a significant number of TUNEL positive cells in 6-24 hours after transient ischemia followed by a decrease at the 48th hour. The number of TUNEL positive cells reached a maximum at the 24th hour after ischemia. The expression of Fas gradually increased as early as when it was at the 6th hour, reached a peak at the 24th hour, and then decreased at the 48th hour. Similarly, the expression of Fas ligand was at peak in 24-48 hours in GCL and INL of retina. Conclusions Retinal ischemia-reperfusion after transient elevated IOP induced apoptosis of cells in the retina. Fas/FasL may play an important role in the early events of the apoptotic pathways. bFGF can rescue RGCs from retinal ischemia/reperfusion injury through downregulation of the expression of Fas/FasL and may represent an important mechanism for therapeutic neuroprotection.

Objective To investigate the effect of hypoxia on expressions of erythropoietin(EPO)mRNA and protein in retinal M?ller cells cultured in vitro. Methods Retina tissues from the new-born Wistar rats were dissected into cell suspension after digested by pancreatin.M?ller cells were separated and purified by mechanical concussion and blowing and striking method.The expression of EPO mRNA and protein under the condition of hypoxia was detected by semi-quantitative reverse transcriptase(RT)-polymerase chain reaction(PCR)and immunocytochemical method. Results Retinal M?ller cells were cultured successfully,95% of which were positively stained by glial fibrillary acidic protein(GFAP).Positively stained EPO protein was located in the cytoplasm and protuberance.The expression of EPO mRNA and protein was faint in the normal retinal M?ller cells,but increased obviously and time-dependently after hypoxia. Conclusion Expression of EPO mRNA and protein increases in M?ller cells after hypoxia,which may be one of the protective factors for the nerves in anoxic retinopathy.

ObjectiveTo investigate the expression of hypoxia inducible factor 1 (HIF-1α) in rats' retinae during the embryonic and earlier postnatal period.MethodsThe retinal expression patterns of HIF-1α protein and mRNA of embryonic day 12 (E12), E16, E20, and postnatal day 1(P1) and P5 rats were determined by immunohistochemical staining and reverse transcription polymerase chain reaction (RT-PCR).ResultsHIF-1α protein was detected in the neural epithelial layer and the pigment epithelial layer at all those 5 timepoints, with higher expression in the ganglion cell layer and the inner plexiform layer, and seems limited to the ganglion cell layer when retina became more mature. Embryonic rat retina had higher expression of HIF-1α protein and mRNA than postnatal retina, the difference was significant (P<0. 01). ConclusionThe expression of HIF-1α in rats' retinae differs from embryonic to earlier postnatal stages.

Aim To investigate the protective effect of erythropoietin(EPO )on neonatal rats'retinal neuronal cells injured by glutamic acid.Methods Retinal neuron cells were cultured and subjected to glutamic acid.The concentration of glutamic acid model was decided by observing the changes of neuronal cells with the releasing quantity of LDH and transmission electron microscope.Then cultured cells were divided into N group(normalcontral),G group(glutamic acid),EG group(EPO and glutamic acid),AG group(AG490 and glutamic acid),and AEG group(AG490,EPO and glutamic acid).MTT assay was applied to detect cell viability in five groups.The expression of Bax was detected with Western blot.Results The changes of mitochondrion and chromatin occurred in 50 ?mol?L-1 group and striking changes occurred in 100 ?mol?L-1 group with transmission electron microscope.There was apparent damage in 20?mol?L-1 group by the releasing quantity of LDH.MTT assay indicated the cell viability of EG group was higher than that of G,AG and AEG group,while,G group cell viability was higher than that of AG and AEG group.The expression of Bax in AG and AEG group was higher than that in G group. And that in G group was higher than in EG group.Conclusions EPO pretreatment can effectively protect the neuronal cells from injuries induced by glutamic acid.Down regulation of Bax and decrease of apoptosis through Jak2 passageway may be the mechanism of protection.

BACKGROUND: Basic fibroblast growth factor (bFGF), a kind of polypeptide growth factor possessing multifunctional biological activities,can protect neurons and promote the growth of nerves. It has been corfirmed that bFGF has therapeutic effects on retina ischemia/reperfusion injury (RIRI).OBJECTIVE: To establish RIRI model and analyze the effects of bFGF on cellular apoptosis of retina and the expression of regulatory gene protein.DESIGN: Randomized grouping and validating trial.SETTING: Department of Ophthalmology, the Affiliated Hospital of Medical College of Qingdao University.MATERIALS: The experiment was conducted at the Research Laboratory of Pathology, Department of Ophthalmology, Medical College of Qingdao University, from April 2002 to December 2003. Twenty-eight healthy Wistar rats were enrolled in this experiment. Four rats were randomly chosen for normal control group, the left eyes of the other 24 rats were set as normal saline control group, and the right eyes were set as bFGF group.METHODS: Normal saline control group and bFGF group adopted the rat RIRI models established by transiently elevating intraocular pressure. Normal saline of 12 μL was injected into the vitreous cavity of the left eyes of the rats in normal control group. 12 μL bFGF was injected into the vitreous cavity of the right eyes of the rats in bFGF group, 4 rats once. No administration was given in normal control group. The expression of apoptotic cells was detected and apoptosis indexes were calculated with the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) method and immunohistochemical staining method at the 1st, 6th,12th, 24th,48th and 72nd hours after reperfusion and ischemia for 1 hour.MAIN OUTCOME MEASURES: ① The detection results of apoptotic cells in situ of retina tissuesat different time points after reperfusion. ②The expression of Fas and caspases-2 in retina tissues at different time points after reperfusion.RESULTS ① Comparison of apoptosis indexes of retina tissues at different time points after ischemia reperfusion: There were no apoptotic cells in the retina tissues of the rats in normal control group. As compared with those in normal saline control group, apoptosis indexes in bFGF group were significantly decreased at ischemia 1 hour and reperfusion 1, 6, 12, 24, 48and 72 hours, especially at the 12th, 24th and 48th hours after reperfusion (t =5.362-5.595, P ＜ 0.05). ② The change of Fas expression at different time points after ischemia reperfusion: There was hardly any Fas expression in normal control group. As compared with that in normal saline control group, Fas expression in bFGF group was significantlydecreased at ischemia 1 hour and reperfusion 1, 6, 12, 24, 48 and 72 hours, especially at the 6th, 12th and 24th hours after reperfusion (t=3.954-9.327, P ＜ 0.05). ③The changes of caspase-2 expression at different time points after ischemia reperfusion: There was no caspase-2 expression in normal control group.Compared with that in normal saline control group, the number of caspase2 positive cells in bFGF group was significantly decreased at the 6th,12th,24th, 48th and 72nd hours after ischemia for 1 hour and reperfusion (t=4.125-15.641, P ＜ 0.05).CONCLUSION: bFGF can significantly inhibit the expression of apoptosis gene Fas and caspase-2 in the ischemia and reperfusion of retina, thus reducing cellular apoptosis of ganglion cells and exerting therapeutic effects on the ischemia and reperfusion of retina.

BACKGROUND: Hypoxia inducible factor-1 α(HIF-1 α) is not only related to physiological reaction of hypoxia,but also takes part in normal embryonic development.OBJECTIVE: To study the alteration of HIF- 1 α in retina during rat development.DESIGN,TIME AND SETTING: Randomized contrast animal study,which was performed in the Shandong Provincial Molecular Virus Key Laboratory,Medical College of Qingdao University between January and September 2007.MATERIALS: Adult Wistar rats,nulliparity,clean grade,and weighing 200 250 g were used in this study.METHODS: Male and female rats were caged as the ratio of 1 : 1.Embryos were obtained at 12-day,16-day,and 20-day pregnancy.Eyeballs were obtained from newborn rats by anesthesia at 1-day,5-day,10-day,and 12-month birth.Retina was separated and made into paraffin section.MAIN OUTCOME MEASURES: Expressions of HIF 1 α protein and HIF-1 α mRNA in retina were measured by immunohistochemistry and semiquantitative reverse transcription-polymerase chain reaction at various dine points of embryonic development.RESULTS: HIF-1 α positively expressed at stratum neuroepitheliale retinae and purpurogenous membrane in the embryonic phase.Additionally,HIF-1 α still positively expressed at stratum neuroepitheliale retinae and purpurogenous membrane,especially in ganglionic cells and inner plexiform layer,in early development.With the gradual development,the positive expression was mainly located at stratum ganglionare retinae.HIF-1 α protein and mRNA expressions were the highest in the embryonic phase,lower in the development,and the lowest in the adult period.There were significantly differences among these three phases (P ＜ 0.01).CONCLUSION: HIF-1 α decreases gradually in retina and its expression is mainly located at stratum ganglionare retinae.

Aim This study aimed to assess the protection of recombinant human erythropoietin (rhEPO) in light-induced injuries in human retinal pigment epithelial(RPE)cells by researching the inhibition of rhEPO for apoptosis in human RPE cells by light-induced injuries.Methods Cultured human RPE cells were exposed to light of 8 w (2 000?500) lux for 12hours,then the culture were stopped at 24 hours after 12hours light stimulation. The effect of inhibiting apoptosis of rhEPO was detected by AnnexinV-flunorescein isothiocyanate/Propidium iodium labeling and flow cytometry. The enzyme linked immunosorbant assay(ELISA)and immunocytochemical staining were used to assess the expressions of caspase-3 and Bcl-2 treated by different doses of rhEPO in light-induced injury on human RPE cells and research the protective mechanism of rhEPO by adding AG490(the special inhibitor of Jak2).Results There was a obviously increased effects on inhibiting apoptosis in every rhEPO group, which was the most conspicuous in 40 IU?ml-1 rhEPO group,and the value was (4.93?1.45)?ml-1. The decrease of expression of caspase-3 was most obvious in 40 IU?ml-1 rhEPO group, and the value was (0.125?0.029) ?g?L-1. The increase of expression of Bcl-2 was the most obvious in 40 IU?ml-1 rhEPO group and the value was 168.21?3.87. But these effects on inhibiting apoptosis in rhEPO group were restrained by adding AG490, the value of apoptosis was (11.29?2.11)?ml-1 and the density of caspase-3 increased to (0.362?0.042) ?g?L-1,the expression of Bcl-2 dropped.Conclusion It is suggested that rhEPO can inhibit the apoptosis of human RPE cells in the light-induced injuries and inhibit the expression of caspase-3 and up-regulate the expression of Bcl-2, so rhEPO can protect the light-induced injuries for human RPE cells. Its protective mechanism is accomplished principally by the pathway of combining EPO with EPOR ,then the combination activates Jak2.

Background The light damage model of retinal pigment epithelium (RPE) cells is a research direction of retinal degeneration diseases,and RPE cell apoptosis induced by light damage and inflammation is an important pathologic basis of light-induced RPE cell damage.However,whether endoplasmic reticulum stress (ERS) paticipates in light-induced RPE cell damage is rarely reported.Objective This study was to explore the effects of ERS on light-induced RPE cell damage.Methods Human RPE cell line (ARPE-19) was cuhured,and light damage models were created by irradiating the cells for 3-,6-,12-and 24-hours with white fluorescent lamp with the intensity of (2 000±500)lx for the selection of optimal irradiating time,and the cells in the normal control group were cultured in the dark environment.The cells were divided into normal control group,light exposure group and 4-phenylb utyric acid (4-PBA) pretreated +light exposure group.The cells from 4-PBA pretreated +light exposure group were cultued firstly with 4-PBA for 30 minutes and followed by light exposure for 12 hours.The apoptisis rate of the cells and intracellular reactive oxygen species (ROS) content were detected by flow cytometry;the concentrations of interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the cell supernatant were assyed by ELISA.The relative expressing levels of activating transcription factor 6 (ATF-6),C/enhancer binding protein homologous protein (CHOP) and caspase-12 mRNA and protein in the cells were detected by real-time quantitative PCR and Western blot,respectively.Results The cultured cells showed a long spindle shape,the border was not clear,the cytoplasm was degranulation,and the cell fragments increased.Flow cytometry showed that compared with the normal control group,the ROS content in the cells and the apoptosis rate were evidently increased with the lapse of light exposure time (F=763.00,119.30,both at P＜0.01).ELISA results showed that the concentrations of IL-1β and TNF-α in the cell supernatant were significantly higher in the light exposure 6-hour group than those in the normal control group with the peak value in the light exposure 12-hour group.Compared with the normal control group,the relative expression levels of ATF-6,CHOP and caspase-12 mRNA and protein in the cells were elevated in the light exposure group and peaked in the light exposure 12-hour group.In addition,the relative expression levels of ATF-6 mRNA,CHOP mRNA and caspase-12 mRNA in the cells were significantly reduced in 4-PBA pretreated+light exposure group compared with the light exposure group (F=281.69,473.88,308.45,all at P＜0.01),and their proteins were also significantly reduced (F =47.86,57.93,106.59,all at P ＜ 0.01).The apoptosis rate,concentrations of IL-1β and TNF-α in the cell supernatant were significantly reduced in 4-PBA pretreated+light exposure group compared with the light exposure group (F =88.64,245.47,101.01,all at P＜0.01).Conclusions The light exposure at (2 000 ± 500)lx induces intracellular ROS accumulation and activates the ERS response,which results in apoptosis and inflammatory process of human RPE cells.4-PBA,a inhibitor of ERS,can suppress light-induced ERS response and therefore reduces the apoptosis rate and inhibits inflammatory process.