Objective To investigate the effect of pigment epitheliumderived factor (PEDF）on the expression of glutamine synthetase in retinal Muuml;ller cells of diabetic rats.Methods Diabetic rats were induced with streptozotocin injection.Before and after injection of 10 mu;l (0.1 mu;g/mu;l) PEDF (experimental group) or 10 mu;l PBS (control group) into the vitreous cavities of diabetic rats respectively for 48 hours,the expressions of GS and IL-1beta; in retina were analyzed by immunohistochemistry and real time RTPCR techniques. After being treated with 100 ng/ml PEDF for 24 hours in high glucose conditions,the expressions of GS and IL-1beta; in cultured Muuml;ller cells were studied by western blot and real time RT-PCR techniques. Apoptosis was analyzed by flow cytometry after Annexin V fluorescein isothiocyanate/Propidium idoium (Annexin V-FITC/PI) staining.Results By immunohistochemistry (the protein level) and real time RT-PCR (the mRNA level),it was found that the expression of GS decreased and the expression of IL-1beta; increased obviously (real time RT-PCR：GS:t=4.23,P＜0.01；IL-1beta;:t=16.73,P＜0.01；immunohistochemistry：GS: t=5.13,P＜0.01；IL-1beta;:t=9.32,P＜0.01) in diabetic rats. After injection of 10 mu;l (0.1 mu;g/mu;l) PEDF into the vitreous cavities of diabetic rats for 48 hours,it was found that the expression of GS increased and the expression of IL-1beta; decreased significantly(RT-PCR GS:t=3.87,P＜0.01IL-1beta;:t=3.61,P＜0.05;immunohistochemistry:GS:t=3.32, P＜0.05；IL-1beta;: t=2.63,P＜0.05). Under high glucose conditions, 100 ng/ml PEDF induced decreasing expression of IL-1beta; and increasing expression of GS significantly (RT-PCR：GS: t=2.89, P＜0.05；IL-1beta;: t=3.37,P＜0.05；Western blot：GS:t=2.66,P＜0.05;IL-1beta;:t=3.23,P＜0.05).Apoptosis of Muuml;ller cells under high glucose conditions was inhibited significantly by the treatment with 100 nmol/ml PEDF (t=3.21,P＜0.05). Conclusions In diabetic rats,PEDF may decrease expression of IL-1beta; in rat retinal Muuml;ller cells, which may result in increasing expression of GS.To some degree,it inhibits possibly the death of retinal ganglion cells.
Objective To observe the regulation effect of transforming growth factor alpha (TGFalpha;) on expression of glutamate transporter（GLAST）and ingestion activity of retinal Muuml;ller cells in mice. Methods To take the retinal tissue of Kunming mouse at postnatal 7～10 day, and then cultured Muuml;ller cells according to literature. The 3～4 generation cultured cells of the same primary cell were divided into two groups at random: ① TGFalpha; group: maintained in different concentrations of TGFalpha; as 50, 75, 125 and 150 ng/ml, 3 holes in each concentration;② Control group: cultured by Eagle culture medium which improved from Dulbeccon and contained 20% fetal calf serum. The influence of different concentrations TGFalpha; on GLAST activity in Muuml;ller cells were observed by L-3H-glutamate uptake detection; the expression of GLAST mRNA in Muuml;ller cells was determined by RT-PCR; the expression of GLAST protein was detected with immunocytochemical staining. Results With the increase of TGFalpha; concentration, both L3H glutamate uptake and GLAST mRNA expression were increased. The L-3H-glutamate accumulation had got to the maximum uptake at concentration of 125 ng/ml, which was 266% of that in control group, meanwhile, the expressions of GLAST mRNA also got to the maximum as 4 times of control group. Immunocytochemical staining indicated that the effect of 125ng/ml TGFalpha; on expression of GLAST protein was higher than that in the control group, the differences between two groups were statistically significant (Plt;0.05). Conclusion TGF-alpha; can increase GLAST activity through up-regulating the expression of GLAST mRNA and protein.
Objective To evaluate the inhibiting effect of adenosine on rat retinal ganglion cells (RGC) death induced by P2X7 and N-methyl-D-aspartate (NMDA) receptor. Methods (1) Long-Evan neonatal rats were back labeled with aminostilbamidine to identify RGC. The viability of RGC affected by P2X7 excitomotor BzATP (50 mu;mol/L), glutamate receptor excitomotor NMDA (100 mu;mol/L) and adenosine (300 mu;mol/L) was detected. (2) RGC from the retinae of unlabeled neonatal rats were cultured in vitro. After labeled with Fura-2 methyl acetate, an intracellular calcium indicator, the effect of BzATP, NMDA and adenosine on intracellular Ca2+ level was detected byCa2+ imaging system. Results Both BzATP (50 mu;mol/L) and NMDA(100 mu;mol/L) could kill about 30% of the RGC. Cell death was prevented by adenosine (300 mu;mol/L) with the cell viability increased from (68.9plusmn;2.3)% and (69.9plusmn;3.2)% to (91.2plusmn;3.5)% (P＜0.001) and (102.1plusmn;3.9)% (P＜0.001), respectively. BzATP (50 mu;mol/L) led to a large, sustained increase of intracellular Ca2+ concentration to (1183plusmn;109) nmol/L. After the adenosine intervened, Ca2+ concentration increased slightly to (314plusmn;64) nmol/L (P＜0.001). Conclusion Adenosine may prevent RGC death and increase of intracellular Ca2+ concentration from P2X7and NMDA receptor stimulation. (Chin J Ocul Fundus Dis, 2007, 23: 133-136)
Objective:To observe the effect of beta;estradiol on gluta mate concentration in rabbitsprime; retinae injured by ischemic reperfusion. Methods:Twenty r abbits ware randomly divided into two groups, the control group and the treatmen t group, with 10 rabbits in each group. Before examined by binocular flash elect roretinography (FERG), retinal ischemic reperfusion (RIR) model was induced in t h e right eyes of all the rabbits by increasing intraocular pressure to 120 mm Hg for 60 minutes; the left eyes were as the control eyes. The rabbits were hypoder mically injected with beta;estradiol (0.1 mg/kg) in treatment group and with phys i ological saline in the control group 2 hours before ischemia. The results of FER G of the right eyes in both of the 2 groups 0, 4, 8, and 24 hours after reperfus ion were record respectively and were compared with the results of FERG before r eperfusion. The retina tissue was collected after the last time of FERG. The con c entration of glutamate was detected by Hitachi L8800 amino acid analyzer. Results:In the right eyes in both of the 2 groups, the result of F ERG showed a beeli ne just after reperfusion. There was no significant difference of awave amplit u de between the 2 groups (t=1.357, 0.798, 0.835; Pgt;0.05); the b wave amplitudes i n experimental group were much higher than those in the control group (t=4.447, 2.188, 3.106; Plt;0.01). The concentration of glutamate in retina was (0.265plusmn;0.014) g/L in the right eyes and (0.207plusmn;0.013) g/L in the left eyes in the control group, and (0.231plusmn;0.007) g/L in the right eyes and (0.203plusmn;0 .014) g/L in the le ft eyes in the treatment group; the difference between the 2 groups was signific ant (F=50.807, P=0.000). There was statistical difference between righ t and left eyes both in the 2 groups and the significant difference of the right eyes betw een the two groups was also found (P=0.000); there was no statistical diffe rence of the left eyes between the 2 groups (P＝0.505). Conclusion:beta;-estradiol may prevent the increase of the concentration of glutamate in retina induced by RIR to protect retinal tissue.
Objective To investigate the expression of induced heat shock protein (HSP) 70 in ratprime;s retinal neurons (RNs) and Muuml;ller cells, and evaluate the protective effect of HSP 70 on RNs injured with glucose deprivation and glutamate. Methods Ratprime;s RNs and Muuml;ller cells cultured in vitro were treated with heat shock (42℃ for 1 hour), and duration of the expression of HSP70 was detected by immunocytochemical techniques. Viability of the cells was measured by methyl thiazolyl tetrazolium (MTT) chromatometry after incitant toxic injury with glucose deprivation (0.56 mmol/L glucose for 6 hours) and glutamate (100 mu;mol/L for 6 hours). Simultaneously, the expression was interdicted by HSP70. Results Hypereffective expression of HSP70 was found in cultured RNs and Muuml;ller cells after heat shock. The viability of RNs pretreated by heat shock after injured with glucose deprivation and glutamate significantly increased which could be interdicted by HSP70 antibody. Conclusion Hypereffective expression of HSP 70 may be induced by heat shock, which enhances the ability of tolerance of RNs to the incitant toxic injury by glucose deprivation and exitotoxicity. (Chin J Ocul Fundus Dis, 2005,21:110-113)
Objective To quantify the mRNA expression of NMDAR1 gene in the retina of eyes with acute elevation of IOP in rabbit． Methods Tweenty-six eyes of 16 rabbits were divided into three groups: Group 1: The IOP of one eye in 10 rabbits was elevated to 60 mm Hg by ante ri or chamber infusion． Group 2: The another eye of the same rabbit in group 1 was maintained the IOP to 20 mm Hg by anterior chamber infusion． Group 3: Unilat eral eyes of six rabbits were enucleated to evaluate the mRNA levels as normal control group． PCR product was identified by Southern blotting and the mRNA expression level was quantified by RT-PCR． Results The results revealed no significant difference between group 1 and group 2． Conclusion This implies that acute elevated IOP may not affect the mRNA expression level of NMDAR1 gene． (Chin J Ocul Fundus Dis, 2001,17:50-51)
Objective To investigate the glutamate toxicity on inner stratum retinal neurons(ISRN) and the neurotoxicity quantity-efficacy relation. Method Retinal explants obtained from 30 neonatal mices were implanted into two pieces of 24-well culture plates (48 wells). The 48 wells were divided into three groups: control group, glutamate exposure 24 h group, and glutamate exposure with further lasting 6 h group. The retinal explants were sectioned, and then stained with HE after 24 h in vitro. The cells in retinal ganglion cells (RGCs) layer and inner nuclear layer (INL) were analyzed by light microscope at 1 000times; magnification , and the number of normal morphological cells was counted under three 1 000times; magnificat ion fields. Results Some cells in ISRN (include RGCs and INL c ells) showed pykno tic nuclei and necrosis after 24 h in control culture. Glutamate exposure 24 h group:at the 2 mmol and 4 mmol concentrations of glutamate, the situation of the normal morphological cells in ISRN had no difference from that of the control group (Pgt;0.05). At the concentration of glutamate more than or equal to 6 mmol, the number of normal morphological cells in ISRN was significantly less than that of the control group (Plt;0.05), and with the increase of glutamate concentration, the number of normal morphological cells was reduced. Glutamate exposure with fur ther lasting 6 h group: at the concentration of glutamate equal to 6 mmol, the n umber of normal morphological cells in INL was significantly less than that of the control group (Plt;0.05), while the number of normal morphological cells in RGCs layer had no difference between two groups (Pgt;0.05). At the concentration of glutamate more than or equal to 8 mmol, the number of normal morphological cels in RGC s layer and INL was significantly less than that of the control group (Plt;0.05 ). Conclusion Glutamate has the neurotoxicity for ISRN in vitro, and the effect is dose-dependant. (Chin J Ocul Fundus Dis, 2001,17:311-314)
Purpose To investigate the roles of vascular endothelial growth factor (VEGF),glutamate and gamma;- aminobutyric acid (GABA) in neovascularization of proliferative diabetic retinopathy (PDR). Methods Vitreous samples were collected from 25 patients (27 eyes)with PDR and 14 patients (14 eyes) with idiopathic macular hole.VEGF levels were determined by ELISA.Amino acids analyses were performed by high-performance liquid chromatography. Results Patients with PDR had significantly higher concentrations of VEGF (median 0.41 ng/ml,quartile 0.54 ng/ml) than controls (median 0.017 ng/ml,quartile 0.01 ng/ml)(Plt;0.001).The levels of glutamate [(11.7plusmn;3.0) mu;mol/L] and GABA [(7.2plusmn;3.9mu;mol/L)] were also higher in patients with PDR than glutamate [(5.8plusmn;0.7) mu;mol/L] and GABA [(3.3plusmn;2.9) mu;mol/L) in controls (Plt;0.05).The glutamate level and GABA level had a significantly positive correlation with VEGF level. Conclusions The increased levels of glutamate and GABA indicate retinal ischemia.The correlations of glutamate and GABA levels with an elevated VEGF level provide biochemical support for ischemi induced neovascularization in patients with PDR.The findings present novel modalities in treatment of PDR. (Chin J Ocul Fundus Dis,2000,16:162-165)