Tetramethylpyrazine Nitrone Protects Retinal Ganglion Cells Against N-methyl-D-aspartate Induced Injury

Purpose: 2-[[(1,1-dimethylethyl)oxidoimino]-methyl]-3,5,6- trimethylpyrazine(TBN) is a derivative of tetramethylpyrazine armed with a nitrone moiety, with dual functions of blocking Ca2+ overload and scavenging free radicals. Here we investigate whether TBN protects retinal ganglion cells(RGCs) against N-methyl-D-aspartic(NMDA) induced injury in SD rat and explore the underlying mechanism.Methods: After intraperitoneal injection of TBN, aqueous humor was extracted at various time points, then TBN concentration in rat aqueous humor was detected by high performance liquid chromatography. Injury of RGCs was induced by intravitreal injection of NMDA or saline as the sham. TBN, or its solvent(negative control), or memantine(positive control) was administrated, i.p. daily for 5 days after the NMDA injection. Retinal functions were tested by electroretinogram(ERG) and visual behavior evaluated by black-white box. The survival rate of RGCs was evaluated by counting Brn3a-positive cells. Expression level of apoptosis related proteins Bax, Bcl-2, cleaved caspase3 and caspase3 was checked by western blotting.Results: TBN quickly entered the aqueous humor and reached a peak concentration around 45 mins after drug administration. TBN significantly increased the survival rate of RGCs. Furthermore, TBN increased the amplitude of photopic negative response to flash, and extended the duration of animals staying in black box after NMDA injury. The elevated ratio of cleaved caspase-3/caspase-3 and bax/bcl-2 induced by NMDA was restored by TBNConclusion: TBN protected RGCs against NMDA injury in rat in both their morphologies and functions. TBN may protect RGCs by inhibiting the apoptotic process. Thus TBN may serve as a promising candidate to treat glaucoma in clinic.