Objective
To investigate the influnce of L-arginine (L-Arg) and L-nitro-arginine-methyl-ester(L-NAME) to purified retinal ganglion cells(RGCs) apoptosis of rats cultured in different consistencies of L-Arg and L-NAME.
Method
RGCs from Sprague Dawley (SD) neonatal rats(postnatal 1~5 day) were cultured in assimilative culture solution in vitro and RGCs were purified by Thy1.1 with sheep anti rat FITC monoclonal antibody. RGCs were cultured in different consistencies of L-Arg and L-NAME: 1×10-6, 1×10-5,1×10-4, 1×10-3, 1×10-2 and 1×10-1 mol/L for 24 hours and 48 hours, respectively. The changes of bcl-2, bax and p53 mRNA in RGCs in different consistencies of L-Arg and L-NAME
were demonstrated qualitatively and quantitatively by in situ hybridization, and their apoptosis were detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL) method, respectively.
Results
After 24 hours in vitro, the purification rate of RGCs in the experiment arrived at 97 %. After 48 hours, there were a few apoptotic cells expression in the control group. Apoptotic cells expression in L-Arg≥1×10-3 mol/L and L-NAME≥1×10-1 mol/L groups increased that had a significant difference with the control group (Plt;0.05). In the group of L-Arg≥1×10-3 mol/L and L-NAME≥1×10-1 mol/L, the expression of bcl-2 mRNA in RGCs became weaker and weaker as the consistencies were increased, but the expression of bax and p53 mRNA in RGCs became higher and higher and had a significant difference with control group (Plt;0.05).
Conclusion
Lower concentration of L-Arg can promote the growth of purified RGCs in vitro and higher concentration of L-Arg can promote the apoptosis of RGCs.
(Chin J Ocul Fundus Dis, 2002, 18: 137-139)
Objective To observe the dynamic expression of nestin and glial fibrilary acidic protein (GFAP) in the development of retina in rats.Methods In 48 Wistar rats, 24 were divided into 8 groups with 3 rats in each according to their age (1 day, 1 week, and 2, 3, 4, 7, 12, and 20 weeks old). The sagittal freezing sections of the eye were made; nestin/glutamine synthetase (GS) and GFAP/GS were stained by immunofluorescence and were observed under the confocal microscope. Total RNA was extracted from 18 rats which were divided into 6 groups according to the age (1 day, 1 week, and 2, 3, 4, and 12 weeks old) with 3 rats in each. The expression of nestin, GAFA and GS mRNA were detected by realtime quantitative reverse transcription polymerase chain reaction (RT-PCR). Müller cells were cultured from postnatal day 7-12 rats; the expression of nestin and GFAP was detected by immunostaining study. Double immunofluorescence was carried out between nestin/GS and GFAP/GS.Results One day after the birth, nestin positive cells were found in the whole retinal neuroblast layers with elongated retinal progenitor cells; the GFAP positive astrocytes were observed in the inner retina. One week after the birth, Müller glial cells expressed GS and nestin but not GFAP; GFAP positive cells localized in the inner retina.Two to 12 weeks after the birth, the expression of nestin in Müller cells decreased and even disappeared; the expression of GFAP in astrocytes didn't change much. The Müller cells expressed nestin but no GFAP in vitro. The expression of nestin and GFAP mRNA in retina was accordant with the results of immunofluorescence staining.Conclusion In the developing retina, the expression of nestin in Müller cells decreases gradually, and no expression of nestin can be found in adult rats; the expression of GFAP can't be observed in Müller cells in neonatal and adult rats.
