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Haiyan Jiang, Xianwei Wang, Shengzhou Wu; Loss-of-function mutation of serine racemase attenuates excitotoxicity by intravitreal injection of N-methyl-D-aspartate . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5450.
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© ARVO (1962-2015); The Authors (2016-present)
A line of evidence suggest that overproduction of D-serine, a racemized product of serine racemase (SR), contributes to neurodegeneration, e.g. in Alzheimer’s disease, amyotrophic lateral sclerosis, epilepsy, diabetic retinopathy. In this study, we aim to clarify the role of SR in excitotoxicity in retina with genetic loss-of-function mutation SR mouse model.
Genotyping, heterozygous mutants carrying the Srrochre269 mutation were obtained from the RIKEN BioResource Center. The progeny were genotyped by PCR amplication combined with endonuclease restriction that detected the absence of an HpyCH4V restriction site in homozygous mutants. Retrograde labeling of RGC, bilateral injection of the superior colliculus were performed in mice (2 mons) under chloral hydrate anesthesia by stereotaxic injection of FAST DiI™ oil, referring to Bregma point with AP -2.92, LAT 0.5, DV at -2 mm. Intravitreal injection of N-methyl-D-aspartate (NMDA), 5 days after the dye infusion, the intravitreal injection of NMDA was performed following pupil dilation using 1% tropicamide and 2.5% phenylephrine after chloral hydrate anesthesia and 1.5 µL, 20 mM NMDA was injected with Hamilton syringe. 24 hrs later, the mice were sacrificed and the RGC were counted under fluorescence microscope. Tunnel staining assay, apoptosis was analyzed with the In Situ Cell Death Detection Kit (Roche) according to the manufacture’s instruction.
Subjected to intravitreal injection of NMDA, the numbers of RGC in mid and posterior segment of retina in srr mutant mice (srr) are 2091+/- 329 (mean +/- SD), 2260+/-375 respectively, significant higher than the numbers in the counterpart, 1655+/-478 (p=0.005), 1862+/-520 (p=0.019) in wild-type mice (w/t). However, the number of RGC in the peripheral segment of retina from srr, 1892+/-602 did not differ significantly from that, 1689+/-429 in w/t (p=0.281). Tunnel staining assay was further used to examine whether the higher RGC number in srr retina is due to the less extent of apoptosis under the induction of NMDA. Statistics: two-tailed Student’ T-test was used comparing to the difference between srr and w/t.
Subjected to intravitreal injection of excitotoxin, NMDA, the retina in w/t contain less amount of RGC than in srr retina. It suggests that srr mutation may provide protection against NMDA excitotoxicity in retina.
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