Change Of Brn3a Expression In Retina After NMDA-induced Excitotoxicity In Rat

Objective To investigate the change of Brn3a expression in retina following NMDA(N-methyl-D-aspartate) excitotoxicity in rat.Methods Thirty adult clean Sprague Dawley rats were divided into 2 group randomly: normal control group had 6 rats, experimental group included 24 rats that right eyes were treated with NMDA by intravitreal injection as experimental eyes, and left eyes were treated with PBS by intravitreal injection as experimental control eyes. Experimental group rats were divided into A, B, C and D group randomly (6 rats included in each group). Four groups of experimental rats were sacrificed at 8,16,24,48h after injection respectively. Changes of Brn3a expression were determined by immuncytochemistry. Semi-quantitative RT-PCR was also used to detect the changes of mRNA of Brn3a in retina.Results 1. Light microscope:The structure of retina in experimental control eyes and normal control eyes were histologically well-defined, uniform in staining, and with regular cell shape.8h after intravitreal injection, vesiculation happened in part of the retinal ganglion cells of experimental eyes.16h later, compared with normal retina, the number of ganglion cells in experimental eyes decreased and the arrangement of cells was loose and irregular. Vesiculation became serious step by step and nuclear condensation was seen in part of ganglion cells. And the thickness of inner nuclear layer decreased.24h after injection, the number of ganglion cells decreased obviously. There was serious nuclear condensation and vesiculation around the nuclear of ganglion cells. The arrangement of inner nuclear cells was even looser and more irregular and the thickness of retina was even thinner than group B. When after 48h, ganglion cell was discovered occasionally and nuclear densification and serious vesiculation was seen in the surviving ganglion cells with irregular shape. The arrangement of the inner nuclear layer cells was gradually more irregular while its thickness decreased.2 Immunohistochemistry:Brn3a expressed in retinal ganglion cell layer only. After 8h of injection, there was no obvious difference on the expression of Brn3a between experimental eyes and the normal eyes. But after 16h. the expression of Brn3a has decreased in experimental retina compared with the normal group. Then 24h later, the number of Brn3a+RGCs decreased obviously in the retina of experimental eyes. And when 48h later, there was little Brn3a+RGC left, which distributed loosely in the experimental retina. There was no obvious difference between experimental control eyes and normal eyes on the expression of Brn3a.3 Real time fluorescence quantitative RT-PCR:8h after injection, the Brn3amRNA expression decreased in experimental eyes compared with normal control eyes, left 78.7% as the normal control group; 16h later, the Brn3amRNA expression decreased obviously, left only 27.1% as the lever of normal control group; 24h after injection, the expression decreased even more seriously, left only 11.5% as the level of the normal control group, when 48h later, the Brn3amRNA expression was left only 6.2% as the lever of normal control group. Meanwhile, there was no obvious difference between the experimental control eyes and the normal eyes on the expression of Brn3amRNA.Conclusion:NMDA can induce excitotoxicity to rat retina, decrease the number of retina ganglion cell and the expression of Brn3a protein as well as its mRNA.