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 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)
ObjectiveTo observe the expression of glutamate (Glu) andγ-aminobutyric acid (GABA) in the retina of diabetic rats which were intervened later by insulin intensive therapy, and to investigate the mechanism of metabolic memory of hyperglycemia which induced the retina neuropathy in diabetic rats. Methods60 Brown Norway rats were randomly divided into normal control (NC) group, diabetes mellitus (DM) group (6 weeks at DM1, 12 weeks at DM2) and metabolic memory (MM) group, 15 rats in each group. Diabetes was induced by intraperitoneal injection of streptozocin. After 6 weeks, MM group was treated with insulin intensive therapy for 6 weeks. DM1 group was sacrificed at the end of 6 weeks and other groups were sacrificed at the end of 12 weeks. High performance liquid chromatography was used to detect the amount of Glu and GABA in the rat retina. Real-time polymerase chain reaction was applied to quantify the mRNA expressions of Glutamate decarboxylase (GAD). TdT mediated dUTP nick ending labelling was used to detect cell apoptosis. ResultsThe concentration of Glu (t=6.963), GABA (t=4.385) and the ratio of Glu/GABA (t=4.163) in MM group were significantly higher than DM1 group, but the concentration of Glu (t=3.411) and GABA (t=3.709) were significantly lower than DM2 group (P < 0.05). And there was no significant difference in the ratio of Glu/GABA between MM and DM2 groups (t=1.199, P > 0.05). The level of expressions of GAD mRNA in MM group was significantly lower than DM1 group (t=3.496, P < 0.05), but higher than DM2 group (t=8.613, P < 0.05). The number of nerve cells apoptosis in MM group was significantly higher than DM1 group (t=2.584, P < 0.05), but lower than DM2 group (t=3.531, P < 0.05). ConclusionsIntensive therapy later by insulin can partially reduce the content of Glu and GABA and the rate of nerve cells apoptosis, which cannot return to normal levels, and has no effect on the rise in the ratio of Glu/GABA caused by the hyperglycemia. The disorders of Glu and GABA may participate in the metabolic memory of hyperglycemia.