Mediation By Mitofusin-2 To NMDA-induced RGCs' Excitotoxicity In Rats

OBJECTIVE:To investigate the mechanism of mediation conducted by mitofusin-2 to NMDA(n-methyl-d-aspartate)-induced retinal ganglion cells(RGCs)excitotoxicity in rats.METHODS :Forty-five adult Sprague Dawley rats were divided into three groups randomly.3ul(40m M,120nmol/l)of NMDA was injected into the vitreous cavity to make excitatory model(with0.1M PBS injection as control).Eyes of each group were removed of lens at 1h,12 h,24h and 48 h after intravitreal injection respectively.Hematoxylin/eosin staining of retinal cross-sections was conducted to observe the morphological alteration.Real-time Fluorescent Quantitative PCR was applied to assess the level of Brn3 a m RNA to measure the degree of injury to RGCs,with level of Mfn2 m RNA detected to analyzed its expression in RGCs.Immunohistochemistry was also used to detect the concentration of Mfn2 expressed in the retina.RESULTS:1.Hematoxylin/eosin staining: Retina of blank control group was in order and uniform dyeing,with vivid structure of cells in every layer.Retina ganglion cells were round or oval which arranged in a single layer.In the NMDA groups,RGCs were hydropic,whose space between the nucleus and cytomembrane was widened.And they were disordered aligned with each other.The number of RGCs decreased and the thickness of inner plexiform layer(IPL)was thinning as the processing time was prolonged,while in PBS injection groups,slight changes were observed.2.Real-time Fluorescent Quantitative PCR of Brn3a: Compared with the blank control group,the expression of Brn3 a m RNA was lower in every group with NMDA injected.nevertheless,there was no statistical significance between PBS injection groups and blank control group(P>0.05).3.Real-time Fluorescent Quantitative PCR of Mfn2: Compared with blank control group,the expression level of retinal Mfn2 in NMDA groups was higher,reached a peak at 24 h after the injection and began to decrease at 48 h.Except the level at 1h after injection(P>0.05),all of groups with NMDA injection were significantly different compared with the blank control group(P<0.05).There was no statistical significance between PBS injection groups and blank control group(P>0.05).4.Immunohistochemistry of Mfn2:the expression of Mfn2 in blank control group and groups with PBS injection was very low in all of the RGCs layer,the inner plexiform layer,outer plexiform layer and the photoreceptor cell layer at which there were trace brownish yellow particles deposited.While Mfn2 expression in RGCs layer was increased in NMDA injection groups.The value of IOD of immunohistochemical results are in good agreement with the RT-PCR's.CONCLUSIONS: Mfn2 may participate in excitotoxicity of RGCs under glaucoma condition,and may be one of the key nodes of oxidative stress caused by mitochondrial dysfunction.