Metallothionein, an Endogenous Antioxidant, Protects against Retinal Neuron Damage in Mice

Abstract

To clarify the functional role of metallothionein (MT) in retinal damage in mice deficient in both MT-I and -II (MT-I/-II-deficient mice [C57BL/6J background]) and wild-type (C57BL/6J) mice and MT induction (zinc sulfate [ZnSO4] and 1alpha, 25-dihydroxyvitamin D3 [Vit. D3]). Retinal, cell damage was induced by intravitreous injection of N-methyl-D-aspartate (NMDA; 40 nmol/eye). Retinal MT-I, -II, and -III mRNA expression was monitored by real-time reverse-transcription-PCR of total retinal RNA from eyes injected or not injected with NMDA. In wild-type mice, MT-I and -II immunohistochemistry was performed (with antibody that recognizes both proteins) 12 and 24 hours after intravitreous NMDA injection. To examine the involvement of induced retinal MT, ZnSO4 (10 nmol/eye) or Vit. D3 (0.2 or 2 ng/eye) was intravitreously injected 24 hours before NMDA injection in wild-type or MT-I/-II-deficient mice, and ganglion cell layer (GCL) cell loss and inner plexiform layer (IPL) thinning were evaluated 7 days after the NMDA injection. The protective effect of Vit. D3 was assessed against the RGC-5 cell death induced by oxidative stress (using buthionine sulfoximine [BSO] to deplete glutathione in combination with glutamate to inhibit cystine uptake). In wild-type mice, MT-II mRNA expression was time-dependently elevated by NMDA (5.9 and 7.4 times versus the nontreated control at 4 and 12 hours, respectively, after injection), with the normal level being regained within 24 hours. In contrast, MT-I and -III showed persistent decreases (to <50% control) from 4 to 24 hours. In wild-type mice, MT-like immunoreactivity was increased in the inner retina (GCL and IPL) 12 and 24 hours after NMDA injection. At 7 days after NMDA injection in MT-I/-II-deficient mice (versus wild-type mice), GCL cell loss was increased, but IPL thickness was not different. Pretreatment with ZnSO4 or Vit. D3 increased inner retinal MT-like immunoreactivity 24 hours after NMDA injection and significantly attenuated NMDA-induced GCL cell loss in wild-type mice, but ZnSO4 pretreatment did not protect against such cell loss in MT-I/-II-deficient mice. In vitro, Vit. D3 pretreatment (100 nM) reduced BSO+glutamate-induced RGC-5 cell death. These findings suggest that MT, especially MT-II, protects against retinal neuron damage, by acting as an endogenous antioxidant.